Supplementary Figure 1: Silibinin has no considerable radiosensitizer effect on

radioresponsive prostate cancer 22RV1 cells. (A) Effect of IR and SB on 22RV1 cell

proliferation after 48 h of treatment. Exponentially growing 22RV1 cells were treated

either with 2.5 or 5 Gy of IR and/or 25 μM SB. After 48 h of these treatments, cells

were collected and total cells were counted using a hemocytometer. (B) Clonogenic

assay was done with 22RV1 cells treated with 2.5 Gy IR alone or in combination with

25 μM SB. The number of colonies with greater than 50 cells were counted in each

group at the end of 10 days. SB, silibinin; n.s., not significant.

Con

trol

25

μM S

B

2.5

Gy

IR +

SB

*

**

B

n.s.

Con

trol

25

μM S

B

2.5

Gy

2.5

Gy

+ SB

5 G

y5

Gy

+ SB

*

*

*

**

A

n.s.

n.s.

Supplementary Figure 2. Silibinin potentiates and prolongs G2/M arrest by IR

exposure in DU145 cells. For cell cycle analysis, DU145 cells were exposed to IR (5

Gy) with or without silibinin (25 SB. After 24 and 48 h of treatments, cells were

processed for saponin/PI staining. Representative histogram showing a prominent

G2/M arrest in combination at 24 and 48 h in DU145 cells. The quantitative data is

shown as figure 2A.

Control 25 M SB 5 Gy IR IR+SB

24 h

48 h

IR+S

B

Con

trol

S

B IR

Con

trol

PC-3DU145

SurvivinPCNA

BIR

+SB

S

B IR

IR+S

B

Con

trol

S

B IR

Con

trol

PC-3DU145

IR+S

B

S

B IR

A

Cyclin B1

Cdc-2

IR+S

B

Con

trol

SB IR

GAPDH

Cdc25C

DU145

Supplementary Figure 3. Silibinin decreases the molecules regulating G2/M phase

transition in PCa cells, but not in HEK-293 cells in response to IR exposure, and

inhibits cell proliferation and survival. (A) mRNA expression levels of G2/M phase

cell cycle regulatory molecules after 48 h of treatment with IR (5 Gy) and /or SB (25

μM) in DU145 and HEK-293 cells. GADPH was used as loading control. (B)

Densitometric quantitation data showing the fold change in the protein levels of PCNA

and survivin (from figure 2D) after indicated treatments in DU145 and PC-3 cells.

GAPDH

Cdc25C

Cyclin B1

Chk-2

IR+S

B

Con

trol

SB IR

HEK-293

48 h

control25 SB5 Gy + 25 SB 5 Gy

DU145

HEK-293

12 h 24 h

48 h 72 h

control

SB

IR+SB IR

Catalase

GST

SOD1

SOD2

GAPDH

IR+

SB

Con

trol

SB IR

A

control

SB

IR+SB IR

control

SB

IR+SB IR

control

SB

IR+SB

IR

B

Supplementary Figure 4. Silibinin enhances IR-induced oxidative stress. (A)

Representative overlap histograms showing ROS levels using DCF-DA treatment of DU145

cells with IR (5 Gy) and/or SB (25 μM silibinin) following 12-72 h. (B) Representative

microscopic pictures showing DCF positive cells (green) in different treatment groups. (C)

RT-PCR analysis of antioxidant enzymes in response to IR and/or SB treatment after 48 h.

(D) Histogram showing ROS levels in DU145 and HEK-293 cells after 24 h of treatment.

C

ControlSB

IR+SB IR

Control SB

IR IR+SB

D

Con

trol

SB

IRIR

+ S

B

Sytox Green pEGFR(Y1068) Merge

3h Post-treatment - JB6 cells

Supplementary Figure 5. Silibinin does not inhibit IR-induced nuclear

translocation of EGFR in JB6 cells. Confocal microscopy showing distribution

of EGFR (red) in JB6 cells in response to IR (5 Gy) and/or SB (25 μM) at 3 h of

treatment. Sytox green (green) was used as nuclear counter stain.

IR

SB IR+SB

Control

GUT: Genitourinary Tract

Mea

n N

eutr

op

hil

C

ou

nt

(K/µ

L)

Mea

n M

on

ocy

te

Co

un

t (K

/µL

)M

ean

RB

C C

ou

nt

(M/u

l)

Mea

n H

b L

evel

(M

/ul)

#

#

$

$

A

B C

D E

Supplementary Figure 6. Silibinin protects normal tissues against radiation injury in

mice. (A) Representative pictures and graph showing discoloration, and shrinkage of the

genitourinary tract (GUT) by radiation treatment which is being rescued by silibinin. (B-E)

Effect of radiation and/or silibinin on (B) mean neutrophil number, (C) mean monocyte

number, (D) mean Hb Levels and (E) mean RBC counts in athymic nude mice after

cumulative 15 Gy (IR) and/or 200 mg/kg body weight of SB treatment.