Chapter-I NGF and/or resveratrol induced differentiation in …shodhganga.inflibnet.ac.in/bitstream/10603/38713/6/chapter-1.pdf · resveratrol in adulthood neurogenesis/ neuro-protection

Chapter-I

NGF and/or resveratrol induced differentiation in PC12 cells

Executive summary

Introduction

Materials and Method

Results

Discussion

NGF and/or Resveratrol induced differentiation in PC12 cells 2014

37

Executive Summary

Therapeutic strategy to stimulate neuronal cell events including proliferation, migration,

differentiation, neurite outgrowth, and synaptogenesis are needed for several

neurodegenerative disorders. However, mechanistic understanding is poor and needs to

be addressed adequately. We investigate the resveratrol (RV) potentiate the action of

NGF to induce neuronal differentiation in PC12 cells. Cytotoxicity assays were used to

identify safe doses of RV. Cells were allowed to differentiate (4 days) under the influence

of selected concentrations of RV. RV induced morphological differentiation was

recorded at an interval of 24 h. Expression (mRNA and protein) studies were carried out

for selected markers of neurogenesis (β-tubulin and NF-M). NGF (50 ng/ml) induced

differentiating cells served as control. Functional interaction of RV with NGF was also

studied by co-exposure of cells to both RV and NGF. The expression changes could be

correlated with the expression of neuronal markers, morphological differentiation and

neurite out growth in the cells. RV shows additive response to NGF induced

differentiation in PC12 cells. Thus, the prophylactic/ therapeutic applicability of

resveratrol in adulthood neurogenesis/ neuro-protection may be suggested.


38

Introduction

Neural differentiation is considered to be a multi-step development process and is

accomplished through the generation of cellular protrusions (Wang, et al., 2007). During

neural differentiation neurite initiation and elongation depends on the formation and

stabilization of integrin-based adhesions (Woo, et al., 2006). These molecules decide the

fate of morphometric changes of cellular protrusion during neural differentiation (Woo, et

al., 2006). It is documented that the impaired neurogenesis may be involved in the

pathophysiology of various brain diseases such as depression, epilepsy, ischemic stroke,

etc (Abdipranoto, et al., 2008; Taupin., 2008). It has also been reported that the gradual

decrease in endogenous NGF production with aging in humans may participate in neuron

degenerative diseases (Haramoto, et al., 2008). It is important to identify biological

modulators that can helpful either in maintaining the production of NGF even with the

increasing age or supplement the function of NGF by inducing neurogenesis. Though, the

studies showing neuronal developmental under the influence of exogenous modulators

are already in place (Gundimeda, et al., 2010; Haramoto, et al., 2008), the signaling

pathways involved with exact cellular and molecular insights of the neurogenesis are

understood poorly and need to be studied to develop better corrective measures.

Resveratrol (RV), a natural polyphenolic compound has shown a wide pharmacological

window with powerful antioxidant activities in both in vitro (Dasgupta et al., 2007) and

in vivo (Della-morte et al., 2009) experimental models of neuronal injuries and neuronal

disorders. Therefore, the present investigation was carried out to understand the cellular

and molecular phenomena involved in RV induced neurogenesis and possible interactions

with NGF.


39

Materials and Method

Culture and maintenance of PC12:

PC12, a rat pheochromocytoma cell line was procured from National Centre for Cell

Sciences, Pune, India, and maintained at In Vitro Toxicology Laboratory, Indian Institute

of Toxicology Research, Lucknow, India. Cells were grown and passaged as per the

standard protocol provided by the supplier. In brief, cells were cultured in Nutrient

Mixture F-12 (Ham), 82.5% supplemented with 2.5% fetal bovine serum (FBS), 15%

horse serum (HS), 0.2% sodium bicarbonate and antibiotic and antimycotic solution

(10X, 1 ml/100 ml of medium, Invitrogen, Life Technologies, USA). The cells were

grown in 5% CO2 - 95% atmosphere in high humidity at 37oC. Prior to use in the

experiments, cells were screened for integrity of established markers (Greene, et al.,

1976; Galbiati, et al., 1998) and viability (Pant, et al., 2001). Batches showing more than

95% cell viability were used in the study. Depending upon the endpoints, cells were

grown in T-25 cm2, T-75 cm2 flasks, 6, 12, 24, 48 and 96 well culture plates.

Identification of non-cytotoxic doses of RV:

Prior starting the expression studies, non-cytotoxic doses of RV were ascertained in PC12

cells using standard endpoints of cytotoxicity including, MTT, NRU, LDH, trypan blue

dye exclusion assays and morphological alterations. The expression studies were carried

out using selected non-cytotoxic doses of RV.

Tetrazolium bromide salt (MTT) assay: MTT assay has been developed by Mosmann

(1983). This assay is based on the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-

diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. This

dye penetrates the cell membranes and passes into the mitochondria where it is reduced to


40

an insoluble, dark purple formazan crystals. The cells are then solubilized with an organic

solvent DMSO. Thus reduction of MTT to the formazan crystals makes it possible to

discriminate between viable or dead cells via spectrophotometric measurements. It was

carried out following the protocol described earlier by Siddiqui et al., (2008). In brief,

cells (1X104) were allowed to adhere in poly-l-lysine coated 96-well culture plates for 24

h under high humid environment in 5% CO2 -95% atmospheric air at 37oC.the medium

was aspirated and cells were subjected to exposure with different concentration of RV in

fresh medium for 24-96 h, Tetrazolium bromide salt (5mg/mL of stock in PBS) was

added 10 µl per well in 100 µl of cell suspension and plate was incubated for 4 h. At the

end of incubation period, the reaction mixture was carefully taken out and 200 µl of

DMSO was added to each well. The plate was kept on rocker shaker for 10 min at room

temperature and then analyzed at absorbance 550 nm using multi well micro plate reader

(Synergy HT, Bio-Tek, USA). Untreated sets were also run under identical condition and

served as basal control.

Neural Red uptake (NRU) assay: NRU assay was done according to method of

Borenfreund E. and Puerner J.A., (1984). NRU assay provides an indication of lysosomal

activity, which is interpreted as a measure of percentage cell viability. The assay was

carried out following the protocol described earlier by Siddiqui et al., (2008). In brief,

cells (1X104) were allowed to adhere in poly-l-lysine coated 96-well culture plates for 24

h under high humid environment in 5% CO2 -95% atmospheric air at 37oC. The medium

was aspirated and cells were subjected to exposure for 24-96 h with different

concentration of RV in fresh medium. After completion of incubation period aspirated all

medium and NRU salt (50µM/mL in medium) was added 100 µl per well plate was

incubated for 3 h. At the end of incubation period, the reaction mixture was carefully


41

taken out, washed with 100 µl washing solution containing 1% CaCl2 (w/v) and 0.5%

HCHO (v/v) to remove the unincorporated dye. Washing solution was removed and

mixture of 200ml 1% acetic acid and 50% ethanol was added. The plate was kept on

rocker shaker for 10 min at room temperature and then analyzed at absorbance 540 nm

using multi well micro plate reader (Synergy HT, Bio-Tek, USA). Untreated sets were

also run under identical condition and served as basal control.

Lactate dehydrogenase (LDH) assay: This assay is based on the cleavage of a

tetrazolium salt when LDH is present in the cell culture medium. Lactate dehydrogenase

(LDH) is a stable cytosolic enzyme, released into the cell culture medium following loss

of membrane integrity resulting from either apoptosis or necrosis. An increase in the loss

of cell membrane integrity during the assay results in an increase of LDH in the culture

medium. Therefore, LDH assay can be used as a marker of cell membrane integrity. It

was carried using the commercially available LDH assay kit for in vitro cytotoxicity

evaluation (TOX-7, Sig St Louis, MO, USA). In brief, following the exposure of RV for

24 to 96 h, culture plate was removed from CO2 incubator as per the experimental

schedule and centrifuged at 250g for 4 min. Then, supernatant of each well was

transferred to a fresh flat bottom 96 well culture plate and further precipitated for

enzymatic analysis as per manufacturer’s instructions.

Trypan blue dye exclusion assays: In principle, the cells with damaged membrane

allow the trypan blue dye to pass into the cytoplasm whereas undamaged cells are

capable of dye exclusion. This dye exclusion method was used to study the cell viability

by assessing the loss of membrane integrity following the method of Pant et al., (2001)

with some modifications. In brief, well-mixed cell suspension (100µl) was added to a test

tube already containing 100 l of trypan blue (0.4%) and the contents were mixed by


42

gentle shaking. In continuation, without wasting the time, 20µl of dye-cell mixture was

placed on the edge of both the chambers of the haemocytometer, prefixed with the cover

slip, allowing the cell suspension to fill the chambers by capillary action. The counting of

unstained (viable cells) and stained cells (nonviable cells) in the four large corner squares

in both counting chambers was made using a 10X microscope objective. The percent cell

viability was calculated by deducting the number of all stained cells from total number of

cells counted (stained cells and unstained cells) over the haemocytometer.

Microscopic examination of cells to study RV induced morphological alterations:

PC12 cells were seeded at a density of 2 x 104 cells / well on poly-l-lysine coated 48-well

plates in culture medium. After 24 h, the cells were exposed to different concentrations of

RV (1–1000 µM). Following the exposures, the cells were observed under inverted

microscope and images were captured at 20X objective (Leica, USA).

NGF and/or RV induced neuronal differentiation in PC12 cells:

Following the declustering, adequate numbers of cells were seeded in poly-L-lysine

(PLL) pre-coated multi well plates and culture flasks (as per the demand of experiment)

and allowed to adhere for 24 h at 37oC in 5% CO2-95% atmosphere under high humid

conditions. Since, PC12 cells are loosely adhered cells and tend to grow in small clusters,

a pre-coating of PLL facilitates the tight adherence of cells to the plastic surface.

Following adherence, medium was aspirated and fresh medium supplemented with RV,

NGF either alone or in combination, was added. Medium was replaced at every alternate

day till the complete maturation of cells by 8 days. PC12 cells were analyzed for

morphological differentiation and expression of marker genes involved in neural

development and differentiation all throughout their maturation (RV/NGF, RV+NGF

group).


43

Morphometric analysis of neurite outgrowth:

Prior strating the expression studies, morphological analysis and quantification of neurite

bearing were carried out using phase-contrast microscope. Briefly, PC12 cells (1X105)

were seeded on poly-L-lysine-coated 6-well plates in the medium with 10% fetal bovine

serum (FBS) for 24 h. The medium was replaced with low serum (1% HS and 0.5% FBS)

containing medium prior the exposure to RV, NGF and combination of both for up to day

8. After incubation, neurite outgrowth of PC12 cell was observed under an inverted

microscope using phase-contrast objectives and photographed by the digital camera. At

least 100 cells in each of ten randomly separated fields were scored and the proportion of

cells with neurites greater than or equal to the length of one cell body was scored positive

for neurite outgrowth, and expressed as percentage of the total cell number in ten fields.

The neurite extension length was also measured for all identified positive neurite-bearing

cells in a field by tracing the longest length of neurite per cell using Image Analysis

Software (Leica Q win-500). The value of neurite length (average maximal neurite length

per neurite-bearing cell in ten fields) was calculated and data from the ten fields in each

well were designated as one experiment. Experiments were repeated at least three times

at each time interval selected in the study (0, 2, 4, and 8 days) and data are expressed as

mean ±SD.

Expression studies in differentiating PC12 cells:

RV induced alterations in the expression of marker genes involved in neural development

both in the presence and absence of NGF were studied in PC12 cells at various stages of

differentiation. Expression changes at mRNA and protein levels were studied using

standard protocols of real-time and western blotting respectively. Immunocytochemical


44

localization and quantification of marker gene products were also done using specific

monoclonal antibodies.

Transcriptional studies (Quantitative Real Time-PCR):

Transcriptional changes were studied using Real Time PCR following exposure of RV

and in the presence and absence of NGF. Briefly, total RNA was isolated from both

experimental and unexposed control sets using Trizol Invitrogen. Total RNA (1µg) was

reverse transcribed into cDNA by superscript 111 first strand cDNA synthesis kit

(Catalog No 18080-05,Invitrogen Lifescience USA). Quantitative Real Time PCR (RT-

PCR) was performed by Syber Green dye (ABI, USA) using ABI PRISM 7900HT

sequence detection system (Applied Biosystem, USA). Real time reactions were

performed in triplicate wells for each sample. The specificity for each primer was

assessed by melting curve analysis and NITCs for respective primers. Actin-β was used

as an internal control to normalize the data. mRNA expression changes are expressed in

relative quantification (RQ).

Translational studies (Western Blot Analysis):

Following respective exposure, cells were washed twice with cold phosphate-buffered

saline, pelleted down and lysed using CelLyticTM M Cell Lysis Reagent (Catalog no.

C2978, Sigma, USA) in the presence of a protein inhibitor cocktail (Catalog no. P8340-

5ML, Sigma, USA), 1mM Na3VO4 and 1mM DTT in blocking buffer (pH 7.5). The

membranes were then re-incubated for 2h at room temperature with secondary anti-

primary immunoglobulin G (IgG)-conjugated with horseradish peroxidase and the blots

were developed using luminol (catalog no. 34080, Thermo Scientific, USA). To

determine the equivalence of protein amounts loaded among different samples, the

developed membranes were stripped with stripping buffer (catalog no. 46430, Thermo


45

Scientific, USA) at 50oC for 30 min, followed by incubation with control antibodies such

as β-actin for further blotting. In some cases, the densitometry for protein specific bands

was done using Gel Documentation System (Alpha Innotech, USA) with the help of

AlphaEaseTM FC StandAlone V. 4.0.0 software. β-actin was used as internal control to

normalize the data. Exposures induced alterations are expressed in relative term fold

change in expression by comparing the data with respective unexposed controls.

Immuno-cytochemical localization studies:

The expression of neuronal marker proteins was also verified with immunocytochemistry

analysis. In this regards, cells were seeded at a density of 10,000 cells/well in PLL coated

8 well chamber slide. After 1 day incubation, the cells were starved with low serum (1%

HS and 0.5% FBS) medium for 16 h and exposed to RV, NGF and combination of both

for 4 days. After completion the exposure, cells were washed with PBS (100 ml/ well)

and then fixed in 4% paraformaldehyde in PBS, pH 7.3 for 1 h at room temperature.

Fixing of cells allows to preserve them in the same state for unlimited time period at 4oC.

For the immunoflorescence staining, the cells were washed with PBS then permeabilized

for 15 minutes with 0.1% Triton X-100 with 2% BSA for 30 minutes. The cells were

incubated with primary antibody such as NF-M and β-III tubulin for overnight at 40C.

The cells were then washed three times with PBS and incubated with secondary FITC or

Rhodamine conjugated monoclonal antibody (Santa Cruz, USA) for 1 h at room

temperature. Following incubation, the cells were washed with PBS and the cells nuclei

were counterstained with 4’-6-diamidino-2-phenylindole (DAPI) with anti-fade

(Invitrogen) mounted with cover slips. Fluorescent labeling was visualized using the

Nikon fluorescent microscope and fluorescence quantification was done using image

analysis software Leica Q-win 500.


46

Statistical analysis:

Results are expressed as mean and standard error of means (mean SE) for at least three

experiments. One-way analysis of variance followed by post hoc Dunnett’s test was

employed to detect differences between the treated and control groups.


47

Results

MTT assay:

Results of percent cell viability of PC12 cells exposed to various concentrations of RV (5

to 1000 μM) for 24-96 h are summarized in Figure-1. The higher concentrations of RV

(100 to 1000 μM) were found to be cytotoxic in dose dependent manner at all the time

points studies. While the concentration (50 μM) was found to cytostatic, as this

concentration seems to be non-lethal and causes a physiological stress only to the cells.

Rest of the lower doses (5 to 25 μM) used in the study were non-cytotoxic.

NRU assay:

Results of percent cell viability of PC12 cells exposed to various concentrations of RV (5

to 1000 μM) for 24-96 h are summarized in Figure-2. The trends of result were as that to

the MTT assay i.e., RV concentrations-100 to 1000 μM was cytotoxic to PC12 cells all

through the exposure periods. Rests of the lower concentrations used were found to be

non-cytotoxic. In general, the magnitude of affect of RV in NRU assay was

comparatively less than MTT assay.

LDH assay:

Highlights of the results of the LDH release assay are summarized in Figure-3. For LDH

release assay, cells were exposed to various concentrations (1-1000 µM) of RV for a

period between 24-96 h. The lower doses of RV (≤ 50 µM) were found to be biological

safe at all the time periods. The concentration dependent increase in the LDH release was

initiated even at 24 h of RV exposure and peaked by 96 h. At all the time interval,

increase in the LDH release levels were concentration dependent and, RV concentrations


48

200, 500 and 1000 µM were found to pose statistically significant effects on LDH release

throughout the exposure period.

Trypan blue dye exclusion assays:

Data highlights of trypan blue dye exclusion assays are summarized in Figure-4. For this

assay also, RV exposure conditions were identical that to MTT, NRU and LDH release

assays. The trends of results were similar to that MTT assay.

Microscopic examination for RV induced morphological alterations in PC12 cells: As

anticipated, the exposure of higher concentration of RV induces morphological

alterations and necrotic cell death. Prior death, cells were found to be spindle shaped with

blabbings, vacuoles and prominent loss of plasma membrane, etc. These detrimental

effects were associated with RV concentration and exposure time. Cells were healthy

upon the exposure of lower concentrations of RV (>50 µM) till the end of exposure

period i.e., 96 h. Among the concentrations used, RV at 10 µM was most effective in

stimulating the neurite outgrowth in PC12 cells (Figure-5).

Neurite outgrowth analysis:

Neurite outgrowth quantification was done to evaluate the neuritogenic action of RV in

our cell system. Cell morphology was observed and percentage of neurite-bearing cells

was counted by phase contrast microscopy. Figure-6 shows that RV has the potential to

induce significant neurite outgrowth (16.58±1.61, 23.07±1.21, and 26.69±0.45 µM) in

significant number of cell population (17.00±1.15, 23±1.15 and 24±1.52) at day 2, 4 and

8 respectively. However, this neurite growth and neurite bearing cell number was

comparatively less than that induced by NGF (27.64±0.89, 35.81±1.24 and 39.89±0.92

µM neurite growth length) and (26±1.73, 32±1.55 and 36±1.71 neurite bearing cell

number) at day 2, 4 and 8 respectively. Interestingly, RV (10 μM) exposure shows


49

additive effect on the neurite outgrowth potential of NGF (50 ng/mL) in cells receiving

the exposure of both RV and NGF. By day 8 of differentiation, the length of neurite

outgrowth values and the number of neurite bearing cells were: 26.69±0.44 μM and

24±1.53 (RV exposure 10 μM), 39.89±0.92 μM and 36±1.73 (NGF exposure 50 ng/mL)

and 45.45±0.68 μM and 43±1.16 (RV 10 μM + NGF 50 ng/mL exposure).

Transcriptional studies (qReal Time-PCR):

Highlights of the results of the real time PCR studies are summarized in Figure-7. A

gradual increase in the upregulation of mRNA expression of neuronal markers viz., NF-

M and β-III tubulin was observed in differentiating cells receiving RV, NGF and

combination of RV + NGF. Cells exposed to RV were showing an increase of 1.46±0.15,

1.59±0.12 fold in NF-M and β-III tubulin respectively at day 2, which finally reached to

2.45±0.2, 1.88±0.16 fold respectively by day 8. The additive effect was found for the

expression of these (NF-M and β-III tubulin) markers in the cells receiving the co

exposure of both RV and NGF viz., 4.20±0.15, 4.93±0.17 fold of control at day 2,

5.97±0.21, 4.43±0.15 fold of control at day 4 and 4.89±0.16, 4.59±0.20 fold of control at

day 8.

Translational studies (Western blot analysis):

To further confirm the neurotrophic effect of RV in presence and absence of NGF,

protein expression of NF-M, β-III-tubulin was determined by western blot analysis and

immunocytochemical localization. In western blot analysis (Figure-8), RV significantly

increases the expression of NF-M (1.45 fold) and β-III-tubulin (1.53 fold) of control

respectively at day 2, which reached to 1.64 and 1.41 fold of control respectively by day

8 of differentiation. In NGF treated cells, the expression of these markers increased by

1.23 and 1.25 fold respectively at day 2 and reached maximum 2.43 and 1.72 respectively


50

at day 8. The expression of these neuronal markers were found to have the additive effect

in cells received co-exposures of RV and NGF at all the points of maturation. At day 2,

the protein expression of NF-M and β-III-tubulin was 2.21 and 2.41 fold of control

respectively, which increased by 2.98 and 2.16 fold at day 4 and finally reached to the

highest levels at day 8 i.e., 3.45 and 2.32 fold of control respectively.

Immunocytochemical localization of marker proteins:

Highlights of the results are summarized in Figure-9. RV significantly increases the

expression of NF-M (1.44±0.12 fold) and β-III-tubulin (1.31±.09 fold) of control

respectively at day 4, while in NGF treated cells, the expression of these markers

increased by 2.36±.098 and 2.17±0.06 fold of control respectively at day 4. As that to

western blot analysis, the co-exposure of RV and NGF show additive effect in up-

regulation of these neuronal markers i.e. 3.57±.011 and 3.14±0.08 fold of control

respectively. DAPI has been used for nucleus staining.


51

Discussion

Neurogenesis induction in progenitor cells in human brain has been suggested as key

therapeutic approach to restore the neurodegenerative disorders. Small molecules may

work as therapeutic agents that raise the high neurotrophic potency and involve in

numerous effects within the brain (Price, et al., 2007). Traditionally, the neuroprotective

effects of polyphenols have been attributed to their ability to exert antioxidant actions

(Ebrahimi, et al., 2012), through their ability to scavenge reactive species, or through

their possible influences on intracellular redox status (Kovacsova, et al., 2010). Recently,

it becomes evident that polyphenols are able to stimulate neuronal regeneration and

induce neurogenesis via their interactions with critical neuronal intracellular signaling

pathways pivotal in controlling neuronal survival and differentiation (Lai, et al., 2011).

RV and related polyphenols are natural compounds found in high concentration in red

wine (Bastianetto, et al. 2000). Recent studies have shown that it could protect from

severe brain injuries and Alzheimer’s disease (Li, et al., 2012). In present investigation,

we have found that RV potentiates the action of NGF to induce neuronal differentiation

in PC12 cells. However, several natural products have been identified that can potentiate

the action of NGF to induce neurite outgrowth in cell culture. But, pharmacokinetic data

for many of these compounds have not been established. Moreover, whether these agents

can cross the blood–brain barrier is not known, and the safety of these agents for humans

has not been determined. Meanwhile, pharmacokinetic data on RV are well established in

humans (Cottart, et al., 2010), and shown to cross the blood–brain barrier (Wendeburg, et

al., 2009). In addition RV has been shown to be safe for humans, and clinical trials are

underway examining the use of these agents in the prevention of cancer (Athar, et al.,


52

2007). As such, RV can be easily and safely evaluated in humans for promotion of

recovery after neuronal injuries as well as for the reversal of age-related loss of neuronal

plasticity. This is the first report showing that RV can potentiate the action of NGF to

induce the morphological and physiological neuronal differentiation in PC12 cells in

eight days time. Cytotoxicity endpoints (MTT, NRU, LDH & Trypan blue assay) were

used to identify safe doses of RV. We also found that exposure of higher concentration of

RV induces morphological alterations and necrotic cell death. Whereas, the cells were

found healthy following the exposure of lower concentration of RV (>50 µM) till the end

of exposure period i.e., 96 h. Among the concentrations used, RV at 10 µM was most

effective in stimulating the neurite outgrowth in PC12 cells (Figure-5). We further

evaluate the neuritogenic action of RV in PC12 cells. RV has the potential to induce

significant neurite outgrowth and in significant number of cell population at day 2, 4 and

8 respectively. However, this neurite growth and neurite bearing cell number was

comparatively less than that induced by NGF day 2, 4 and 8 respectively. Interestingly,

RV (10 μM) exposure shows additive effect on the neurite outgrowth potential of NGF

(50 ng/mL) in cells receiving the exposure of both RV and NGF. We further confirmed

the neurotrophic effect of RV in presence and absence of NGF through expressional

studied of neuronal markers viz NF-M, β-III-tubulin. The neuratogenic capabilities of RV

were found comparatively lower than NGF (50 nM). However, a co-exposure of RV (10

μM) + NGF (50 nM) was found to have an additive effect on the upregulation of

expression (mRNA and protein) of neuronal markers. The expressional changes could be

correlated with the expression of neuronal markers (NF-M, β-III-tubulin) in the cells.

Resveratrol shows additive response to NGF induced differentiation in PC12 cells. Thus,


53

the NGF-potentiating activity of RV may be a highly useful tool to maintenance of brain

function defects of memory, and aging of the brain.

Figure-1: Mitochondrial activity by MTT assay in PC 12 cells following the

exposure of RV (1−1000 μM) at various time interval (24−96h). PC12 cells were

seeded on poly-L-lysine-coated 96 well plate for 24 h prior to exposure to different

concentrations (1−1000 μM) of RV for 24−96 h and MTT assay was subsequently

performed. Values are mean ± SEM of three experiments each carried out in triplicate

and compared as indicated in the figure by Dunnett’s test (*P < 0.05, **P < 0.01).

* p

Figure-2: Lysosomal activity by NRU assay in PC 12 cells following the exposure

of RV (1−1000 μM) at various time interval (24−96h). PC12 cells were seeded on

poly-L-lysine-coated 96 well plate for 24 h prior to exposure to different

concentrations (1−1000 μM) of RV for 24−96 h and NRU assay was subsequently

performed. Values are mean ± SEM of three experiments each carried out in triplicate

and compared as indicated in the figure by Dunnett’s test (*P < 0.05, **P < 0.01).

* p

Figure-4: LDH assay in PC 12 cells following the exposure of RV (1−1000 μM) at

various time interval (24−96h). PC12 cells were seeded on poly-L-lysine-coated 96

well plate for 24 h prior to exposure to different concentrations (1−1000 μM) of RV for

24−96 h and LDH assay was subsequently performed. Values are mean ± SEM of three

experiments each carried out in triplicate and compared as indicated in the figure by

Dunnett’s test (*P < 0.05, **P < 0.01).

* p

Figure-4: Trypan blue assay in PC 12 cells following the exposure of RV (1−1000

μM) at various time interval (24−96h). PC12 cells were seeded on poly-L-lysine-

coated 96 well plate for 24 h prior to exposure to different concentrations (1−1000

μM) of RV for 24−96 h and Trypan blue assay was subsequently performed. Values are

mean ± SEM of three experiments each carried out in triplicate and compared as

indicated in the figure by Dunnett’s test (*P < 0.05, **P < 0.01).

* p

Figure-5: Microscopic examination of PC 12 cells following various doses of

RV. PC12 cells were seeded on poly-L-lysine-coated 6 well plate for 24 h prior to

exposure to different concentrations (1−1000 μM) of RV for 24 h and check the

effect of various doses of RV in PC12 cells. Images were captured via phase

contrast microscope.

Control

RV-5 µMRV-25 µM RV-10 µM

RV-50 µMRV-100 µM

RV-1000 µM RV-500 µM

RV-200 µM

Figure-6: Neurite Bearing and elongation measurement in PC12 cells at day 4

induced by RV or/and NGF Neurite outgrowth was determined as indicated in

materials and methods. The results are shown as the mean ± SE for three

experiments. The values obtained were compared as indicated in the figure by

Dunnett’s test (*P < 0.05, **P < 0.01). (*P < 0.05, **P < 0.01).

CONTROL RV10µM

RV10µM+NGF-50ng/mL

NGF-50ng/mL

Number of neutrites bearing cells in PC12 cells

Nu

mb

er

of

ne

utr

ite

s b

ea

rin

g c

ell

s

Concentrations

0

5

10

15

20

25

30

35

40

45

50Day2 Day4 Day8

*

* ***

****

**

****

* p

Figure-7: mRNA expression profiling of neuronal markers genes i.e. β-III

tubulin and NF-M in PC12 cells at various day of maturity from day2 to day 8

induced by RV or/ and NGF. PC12 cells were seeded on poly-L-lysine-coated 25

cm2 flask in normal medium for 24 h and then shifted to low serum medium (1%

HS and 0.5% FBS) for 24 h prior to exposure to RV (10 µM) or/ and NGF (50

ng/mL) from Day 2 to Day8 and check the expression of neuronal markers genes by

Real-time PCR. β-actin was used as internal control to normalize the data. The

values obtained were compared as indicated in the figure by Dunnett’s test (*P <

0.05, **P < 0.01).

0

1

2

3

4

5

6

7 Day2 Day4 Day8

NF-M

**

****

**

**

**

**

**

0

1

2

3

4

5

6Day2 Day4 Day8

β-III tubulin

***

**

**

** **

****

**

* p

Figure-8 :Protein expression profiling of neuronal markers genes i.e NF-M and

β-III tubulin in PC12 cells at various day of maturity from day2 to day8

induced by RV or/ and NGF. PC12 cells were seeded on poly-L-lysine-coated 25

cm2 flask in normal medium for 24 h and then shifted to low serum medium (1%

HS and 0.5% FBS) for 24 h prior to exposure to RV (10 µM) or/ and NGF (50

ng/mL) from Day 2 to Day8 and expression of neuronal markers genes were

checked by western blotting. β-actin was used as internal control to normalize the

data.

1 1.45 1.67 2.21

1 1.23 1.54 2.98

1 1.64 2.43 3.45

Day-2

C RV NGF RV+NGF

NF-M

Day-4

Day-8

β-actin

NF-M

β-actin

NF-M

β-actin

1 1.53 1.89 2.43

1 1.25 1.58 2.16

1 1.41 1.72 2.32

β-tubulin

C RV NGF RV+NGF

β-tubulin

β-tubulin

β-actin

β-actin

β-actin

Day-2

Day-4

Day-8

Control

RV- 10μM

NGF- 50ng

RV+NGF

β-III tubulin NF-M MergeDAPI

Figure-9: Immunocytochemical localization of neuronal markers genes i.e β-III

tubulin and NF-M in PC12 cells at day 4 induced by RV or/and NGF. PC12

cells were seeded on poly-L-lysine-coated 25 cm2 flask in normal medium for 24 h

and then shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior to

exposure to RV (10 µM) or/ and NGF (50 ng/mL) for Day 4 and check the

expression of neuronal markers genes by immunocytochemical localization. Images

were captured via a Nikon phase contrast fluorescence microscope (model 80i) with

an attached 12.7 megapixel Nikon DS-Ri1 digital CCD cool camera. Quantification

of fluorescence was done using image analysis software Leica Q-win 500, and data

expressed in-fold of unexposed control. The results are shown as the mean ± SE for

three experiments and compared as indicated in the figure by Dunnett’s test (*P <

0.05, **P < 0.01).

0

0.5

1

1.5

2

2.5

3

3.5

4

Control RV NGF RV+NGF

NF-M β-III Tunulin

**

**

**

*

* p

Documents

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