ULEMENTARY INORMATION - static-content.springer.com10.1038/ncb3192... · of the introduced stop codon (C terminus). Data are presented as mean ± SD (n=3 independent experiments,

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DOI: 10.1038/ncb3192

Beclin1

VPS34

UVRAG

ATG14

Rubicon

p22 PHOX

p-p40PHOX

ATG7

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LC3-II

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Supplementary Figure 1: LAP is a pathway distinct from canonical autophagy.

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Supplementary Figure 1 LAP is a pathway distinct from canonical autophagy. (a) RAW cells were fed inert beads or Pam3csk4-beads for 1 hour. Immunofluorescent staining was performed for LC3B and analyzed by microscopy. Representative images are shown (above), and percent of LC3+ phagosomes is calculated (below). Data are presented as mean ± SD (n=50 cells per genotype pooled from two independent experiments, **p,0.001 using Student’s t test). (b) RAW cells were allowed to phagocytose inert beads or Pam3csk4-beads for 1 hr. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (c) RAW-GFP-LC3 cells

were fed inert beads or Alexa Fluor 594-zymosan, and internalization and GFP-LC3 translocation were followed at 4-min intervals for 6 h. Representative images from three independent experiments are shown (top). Time (in minutes) is indicated above each panel. Time course of GFP-LC3 translocation to the inert bead- or zymosan-containing phagosome. Data are presented as mean ± SD (n=75 cells per genotype pooled from three independent experiments, **p,0.001 using Student’s t test). (d) RAW-GFP-LC3 cells were fed inert beads or Alexa Fluor 594-zymosan for 1 hour. Whole cell lysates from pre-digitonin treated samples (right), and digitonin-treated samples (left) were solubilized in SDS-PAGE and blotted for LC3B. The results presented are representative of two independent experiments.

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Supplementary Figure 2 Generation of the Rubicon-deficient mouse model using the CRISP/Cas9 method. (a) Western blot analysis of organs from Rubicon+/+ and Rubicon-/- mice, aged 8 weeks. Representative images from three independent experiments are shown. (b) Relative mRNA level of Rubicon of organs from Rubicon+/+ and Rubicon-/- mice (aged 8 weeks) was quantified by real-time PCR. Data normalized to actin. Primers were designed to cover a region upstream of the introduced stop codon (N terminus) and downstream of the introduced stop codon (C terminus). Data are presented as mean ± SD (n=3 independent experiments, **p < 0.001 using Student’s t test; in each experiment, technical triplicate samples of 3 mice per genotype were assessed). (c) Expected and observed frequency of offspring from Rubicon+/- x Rubicon+/- crosses of mice. All genotypes were observed at Mendelian ratios. (d-f) FACS analysis of immune cell composition in the spleen (d), lymph node (e), and thymus (f) of Rubicon+/+, Rubicon+/-, and Rubicon-/- mice, aged 12

weeks. Data are presented as mean ± SD (n=3 independent experiments; in each experiment, technical triplicate samples of 100,000 cells each per genotype were assessed). (g) Rubicon+/+ GFP-LC3+ and Rubicon−/− GFP-LC3+ bone marrow-derived macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) was quantified by flow cytometry. Data are presented as mean ± SD (n=3 independent experiments; in each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed). (h) RAW cells were transfected with Scrambled (Scr.) or RAB7 siRNA oligonucleotides. After 48 hours of transfection, cells were fed Pam3csk4-beads for 1 hr. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. Representative images from two independent experiments are shown.

SpleenLiver

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Expected Observed Rubicon+/+ 50 (25%) 58 (29%) Rubicon+/- 100 (50%) 99 (49.5%) Rubicon-/- 50 (25%) 43 (21.5%) Total 200 200

Supplementary Figure 2: Generation of the Rubicon-deficient mouse model using the CRISPR/Cas9 method.

Rubicon+/+Rubicon+/-Rubicon-/-d

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Rubicon+/+ Rubicon+/+Rubicon-/-Rubicon-/-

N terminus C terminus

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NK1.1+ B220+ CD3+ CD4+

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Beclin1

VPS34

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LC3-II

Cre- FIP200f/f

+Pam3csk4-bead Cre+

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Beclin1

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+Pam3csk4 Bead d

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Supplementary Figure 3: LAP occurs independently of the pre-initiation complex.

c

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Supplementary Figure 3 LAP occurs independently of the pre-initiation complex. (a-b) Bone marrow-derived macrophages from LysM-Cre- FIP200flox/flox GFP-LC3+ and LysM-Cre+ FIP200flox/flox GFP-LC3+ mice were left untreated (NS) or were cultured with 200 nM rapamycin (Rapa., a), starvation conditions (S, b), Inert beads (I, b), or Alexa Fluor 594-zymosan (Zymosan or Z, a-b). (c) Bone marrow-derived macrophages from LysM-Cre- FIP200flox/flox GFP-LC3+ and LysM-Cre+ FIP200flox/flox GFP-LC3+ mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 hr. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (d) Bone marrow-derived macrophages from ULK1+/+ and ULK1-/- mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 hr. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome

proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (e-h) RAW-GFP-LC3 cells were transfected with Scrambled, Ambra1, or WIPI2 siRNA oligonucleotides. After 48 hours of transfection, cells were left untreated (NS) or were cultured with 200 nM rapamycin (Rapa., e), starvation conditions (S, f), Inert beads (I, f), or Alexa Fluor 594-zymosan (Zymosan or Z, e-f). GFP-LC3 puncta was assessed at 18 h, and translocation of GFP-LC3 to the LAPosome was assessed at 1 h by confocal microscopy (e) and flow cytometry (f). Whole cell lysates were analyzed for deletion efficiency of Ambra1- (g) or WIPI2-siRNA-treated (h) RAW-GFP-LC3 cells. For (a), (c), (d), and (e), representative images from three independent experiments are shown. For (b) and (f), Data are presented as mean ± SD (n=3 independent experiments, *p<0.05, **p < 0.001 using Student’s t test; for each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS).




Supplementary Figure 4: LAP utilizes a UVRAG-containing Class III PI3K complex.

Beclin1 Actin

Cre: Beclin1flox/flox

− +


LysM-Cre+ Beclin1flox/floxCre- Beclin1f/f

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Supplementary Figure 4 LAP utilizes a UVRAG-containing Class III PI3K Complex. (a-b) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre- Beclin1flox/flox and LysM-Cre+ Beclin1flox/flox macrophages (a). Macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (b). (c-d) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre- VPS34flox/flox and LysM-Cre+ VPS34flox/flox macrophages (c). Macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (d). (e-f) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre- ATG14flox/flox and LysM-Cre+ ATG14flox/flox macrophages (e). Macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were

quantified by flow cytometry (f). (g-h) Whole cell lysates were analyzed for deletion efficiency of Scrambled siRNA- and UVRAG siRNA-treated RAW-GFP-LC3 cells (g). Macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (h). (i-j) Bone marrow-derived macrophages from wild-type, Rubicon-/-, or NOX2-/- mice were fed inert beads or Pam3csk4-beads for 1 hour. Immunofluorescent staining was performed for the proteins indicated above each panel and analyzed by microscopy. Representative images (i) and signal intensity profiles (j) for Beclin1, UVRAG, and VPS34 across phagocytosed beads are quantified. Data (intensity measurements across beads) are presented as mean ± SD (n=50 cells per genotype per stain pooled from two independent experiments). For (b), (d), (f) and (h), data are presented as mean ± SD (n=3 independent experiments; technical triplicate samples of 50,000 cells each per genotype per experiment were assessed).




Supplementary Figure 5: NOX2 is downstream of the Class III PI3K complex and required for LAP

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Cre+ Beclin1f/f Cre- Beclin1f/f

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Supplementary Figure 5 NOX2 is downstream of the Class III PI3K Complex and required for LAP. (a) NOX2+/+ GFP-LC3+ and NOX2−/− GFP-LC3+ bone marrow-derived macrophages were fed Alexa Fluor 594-zymosan, and the percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry. (b) Bone marrow-derived macrophages from genetic knockout strains were fed inert beads or Alexa Fluor 594-zymosan and analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). Filled grey histogram represents inert bead. Representative plots from three independent experiments are shown. (c) NOX2+/+ and NOX2−/− bone marrow-derived macrophages were fed Pam3csk4-beads (30 minutes). mVPS34

was immunoprecipitated from the purified LAPosomes and used in the Class III PI3K Activity assay. Data are presented as pM of PI(3)P,. (d) Bone marrow-derived macrophages from WT, LysM-Cre+ Beclin1flox/flox, and ULK1-

/- mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 hr. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. For (a) and (c), data are presented as mean ± SD (n=3 independent experiments; for each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS).




Supplementary Figure 6: The activity of Rubicon and NOX2 are required for translocation of the downstream conjugation systems to the LAPosome.

WT

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/- W

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Supplementary Figure 6 The activity of Rubicon and NOX2 are required for the translocation of downstream conjugation systems to the LAPosome. (a) Bone marrow-derived macrophages from wild-type, Rubicon-/-, or NOX2-/- mice were fed inert beads or Pam3csk4-beads for 1 hour. Immunofluorescent staining was performed for the proteins indicated above each panel and analyzed by microscopy. Representative images from two independent experiments are shown. (b) Model of proposed activity of Rubicon in the crosstalk of Class III PI3K and NOX2 complexes. Recruitment of the Rubicon- and UVRAG-containing Class III PI3K

complex allows for sustained VPS34 activity at the LAPosome, resulting in significant PI(3)P deposition on the LAPosome membrane. This PI(3)P allows for the recruitment of autophagic downstream conjugation systems to the LAPosome and stabilizes the NOX2 complex via its binding to p-p40PHOX. Rubicon itself also stabilizes the NOX2 complex, promoting optimal ROS production. Rubicon mediates the crosstalk between the Class III PI3K and NOX2 complexes, resulting in lipid oxidation and PI(3)P generation, both required for conjugation of LC3 to the lipids of the LAPosome.




Supplementary Figure 7: PI(3)P and ROS are both required for LAP

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Supplementary Figure 7 PI(3)P and ROS are both required for LAP. (a) Bone marrow-derived macrophages from genetic knockout strains were fed Pam3csk4-beads for 30 minutes. Immunofluorescent staining was performed for oxidized LDL (OxLDL) and PI(3)P and analyzed by confocal microscopy. Representative images (left) and signal intensity profiles for OxLDL (middle) and PI(3)P (left) across phagocytosed beads are quantified. Data (intensity measurements across beads) are presented as mean ± SD (n=60 cells per genotype per stain pooled from two independent experiments). (b) RAW-GFP-LC3 cells were fed inert beads or zymosan, in the presence or absence of tert-butyl hydroperoxide (TBHP, 100 μM, 50 μM), Tiron (1 mM, 0.5 mM), or 3-MA (25 mM, 5 mM). Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). (c) RAW-GFP-LC3 cells were fed inert beads or Pam3csk4-beads, in the presence or

absence of Tiron (1 mM, 0.5 mM) or 3-MA (25 mM, 5 mM). After 1 hour of phagocytosis, phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of four independent experiments. (d) RAW cells were fed inert beads, zymosan, or HRP-coupled beads. Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE (e) RAW cells were fed inert beads (I), Pam3csk4-beads (P), Catalase-beads (C), or Pam3csk4+Catalase –beads (PC). Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). For (b), (d), and (e), data are presented as mean ± SD (n=3 independent experiments, **p < 0.001 using Student’s t test). For each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS.




Supplementary Figure 8 Clearance of Aspergillus fumigatus requires LAP. (a-b) Bone marrow-derived macrophages from GFP-LC3+ genetic knockout strains were fed inert beads or A. fumigatus-dsRed and analyzed by flow cytometry for GFP-LC3 translocation to the LAPosome (a) and phagocytosis (b). The percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed A. fumigatus, right) were quantified by flow cytometry. Data are presented as the average of two independent experiments. (c) Bone marrow-derived macrophages from different genetic knockout strains were fed live A. fumigatus at an MOI of 1, and percentage of killing was calculated as [total

cfu at time-point \ cfu at infection]. (d-e) Mice of different genetic knockout strains (5 mice/strain) were infected intranasally with live A. fumigatus conidia. Percent weight loss was monitored at days 3 and 7 post-infection (d). Serum was collected at day 7 post-infection and analyzed for cytokines via Luminex technology (e). In (c), (d), and (e), data are presented as mean ± SD (n=3 independent experiments, *p<0.05, **p<0.001 using Student’s t test. In (c), technical triplicate samples of each genotype per timepoint per experiment were assessed. In (d) and (e), technical triplicate samples from 5 mice per genotype per timepoint per experiment were assessed.




Supplementary Figure 9: Western blot scans

Rubicon

ATG7

LC3-II

UNC93B

Beclin1

VPS34

UVRAG

Figure 1e

Rubicon

VPS34

Beclin1

UVRAG

UNC93B

LC3-II

Figure 1f

Supplementary Figure 9 Western blot scans. Phagosome purification samples were prepared as described in Methods. The entire phagosome purification was run on 1 – 2 SDS-PAGE gels due to the relatively lower protein yield compared to whole-cell lysate samples. Membranes were sectioned according to the molecular weight marker, and proteins residing within that range of molecular weights were probed with antibodies listed above. When necessary, membranes were stripped with Restore PLUS

Western Blot Stripping Buffer (Life Technologies), re-blocked in 1X TBST with 5% w/v nonfat dry milk, and probed with fresh antibodies. Images were captured with an Amersham Imager 600 and saved as tiff files. The western images shown are the specific sections that we probed to allow for the analysis of multiple proteins from a limited sample volume. All the original unedited images from key experiments are included in this supplementary figure and are representative of reproducible, independent experiments.





UNC93B

Beclin1

p-p40 PHOX

p22 PHOX

LC3-II

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Figure 3f

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Rab5

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Figure 2e

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Figure 3g

Supplementary Figure 9 continued




Figure 6g

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Figure 5a


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p-p40 PHOX

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Figure 4f Figure 3h






Figure S2a

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Figure S2h

Rubicon

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Figure S3c






Beclin1

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Figure S7c

Figure S3d





Protein DescriptionLog2

(IgG-bead / Inert-bead)

Log2 (Pam3csk4-bead /

Inert-bead)

Log2 (Avg)

Rubicon Run domain Beclin-1 interacting and cysteine-rich containing protein 1.21 1.35 1.28

Prkcd Protein kinase C δ 1.91 2.63 2.23

Lgals3 Galectin-3 3.21 2.93 3.07

Ifitm3 Interferon-induced transmembrane protein 2 2.36 1.66 2.01

Srrm2 Serine/arginine repetitive matrix protein 2 2.19 1.85 2.02

Myof Myoferlin 3.13 2.68 2.89

02-Sep Septin-2 1.55 1.92 1.995

Ap2s1 AP-2 complex subunit ς 1 2.63 2.25 2.44

Trim33 E3 ubiquitin-protein ligase 2.58 1.69 2.135

Xrcc5 X-ray repair cross-complementing protein 5 1.03 5.98 3.505

Lalba Lactalbumin-α 5.23 1.39 3.31

Saa3 Serum amyloid A-3 3.43 2.54 2.985

Hck Tyrosine-protein kinase HCK 2.28 1.81 2.045

Tmem206 Transmembrane protein 206 1.71 2.44 2.075

Snx20 Sorting nexin-20 2.37 2.83 2.6

Atp8b3 ATPase, class I, type 8B, member 3 0.96 0.81 2

Reps1 RalBP1-associated Eps domain-containing protein 1 4.87 1.29 3.08

Gabarap Gamma-aminobutyric acid receptor-associated protein 2.04 2.33 2.185

Gabarapl1 Gamma-aminobutyric acid receptor-associated protein-like 1 1.17 1.77 1.47

Map1lc3b Microtubule-associated proteins 1A/1B light chain 3B 0.91 0.71 0.81

Pik3c3 Phosphatidylinositol 3-kinase catalytic subunit type 3 1.09 1.17 1.13

Atg3 Ubiquitin-like-conjugating enzyme ATG3 N/A 0.72 0.72

Atg9 Autophagy 9-like 1 protein 0.89 0.53 0.71

Name MM* PAM Chr Start End Strand Location Genestarget 0 NGG chr16 32847302 32847324 - exonic 1700021K19Rik (Rubicon)Off01 2 NGG chrX 156475145 156475167 - intronic Map3k15Off02 3 NGG chr11 16609070 16609092 + intergenic Sec61g,EgfrOff03 3 NGG chr2 30122383 30122405 - exonic Phyhd1Off04 3 NAG chrX 13213424 13213446 - intronic Cask,Gpr34Off05 3 NAG chrX 11686178 11686200 - intronic BcorOff06 3 NAG chr16 7079230 7079252 - intronic Rbfox1Off07 3 NGG chr7 151526315 151526337 - intronic Shank2Off08 3 NGG chr9 49739346 49739368 - intergenic Ncam1, 2310014F07RikOff09 3 NAG chr1 58567252 58567274 + intergenic Orc2, Fam126bOff10 3 NGG chr10 109322284 109322306 + intronic Nav3Off11 3 NAG chr6 91915442 91915464 - intergenic 4930590J08Rik, Fgd5Off12 3 NGG chr19 58049718 58049740 - intronic Atrnl1Off13 3 NAG chr15 83055133 83055155 + intergenic Cyb5r3, A4galtOff14 6 NGG chr11 73029624 73029646 - intronic Shpk Off15 6 NAG chr3 118690448 118690470 + intronic DpydOff16 6 NAG chr5 67173306 67173328 - intronic Limch1Off17 6 NAG chr8 37646560 37646582 - intronic Dlc1Off18 7 NGG chr14 85943851 85943873 + intergenic Pcdh17, Diap3Off19 4 NGG chr4 81475756 81475778 + intergenic Mpdz, NfibOff20 2+ 1bp del NAG chr1 9344457 9344457 - intergenic Sntg1, Rrs1Off21 2+ 1bp ins NAG chr2 26861506 26861506 + intronic Adamts13

*MM, Number of mismatches in the 20mer target sequence; Chr, chromosome; Start and End, begin and end of the coordinates of the sites (NCBI37/mm9 mouse assembly).

Supplementary Table 1 Proteins uniquely associated with the LAPosome. Proteins associated with the LAPosome with a log2 SILAC ratio greater than 0.7 to the inert bead phagosome. The protein name, description, Log2 ratios for both IgG-bead/inert bead and Pam3csk4-bead/inert beads, and the average Log2 ratio are shown.




Protein DescriptionLog2

(IgG-bead / Inert-bead)

Log2 (Pam3csk4-bead /

Inert-bead)

Log2 (Avg)

Rubicon Run domain Beclin-1 interacting and cysteine-rich containing protein 1.21 1.35 1.28

Prkcd Protein kinase C δ 1.91 2.63 2.23

Lgals3 Galectin-3 3.21 2.93 3.07

Ifitm3 Interferon-induced transmembrane protein 2 2.36 1.66 2.01

Srrm2 Serine/arginine repetitive matrix protein 2 2.19 1.85 2.02

Myof Myoferlin 3.13 2.68 2.89

02-Sep Septin-2 1.55 1.92 1.995

Ap2s1 AP-2 complex subunit ς 1 2.63 2.25 2.44

Trim33 E3 ubiquitin-protein ligase 2.58 1.69 2.135

Xrcc5 X-ray repair cross-complementing protein 5 1.03 5.98 3.505

Lalba Lactalbumin-α 5.23 1.39 3.31

Saa3 Serum amyloid A-3 3.43 2.54 2.985

Hck Tyrosine-protein kinase HCK 2.28 1.81 2.045

Tmem206 Transmembrane protein 206 1.71 2.44 2.075

Snx20 Sorting nexin-20 2.37 2.83 2.6

Atp8b3 ATPase, class I, type 8B, member 3 0.96 0.81 2

Reps1 RalBP1-associated Eps domain-containing protein 1 4.87 1.29 3.08

Gabarap Gamma-aminobutyric acid receptor-associated protein 2.04 2.33 2.185

Gabarapl1 Gamma-aminobutyric acid receptor-associated protein-like 1 1.17 1.77 1.47

Map1lc3b Microtubule-associated proteins 1A/1B light chain 3B 0.91 0.71 0.81

Pik3c3 Phosphatidylinositol 3-kinase catalytic subunit type 3 1.09 1.17 1.13

Atg3 Ubiquitin-like-conjugating enzyme ATG3 N/A 0.72 0.72

Atg9 Autophagy 9-like 1 protein 0.89 0.53 0.71

Name MM* PAM Chr Start End Strand Location Genestarget 0 NGG chr16 32847302 32847324 - exonic 1700021K19Rik (Rubicon)Off01 2 NGG chrX 156475145 156475167 - intronic Map3k15Off02 3 NGG chr11 16609070 16609092 + intergenic Sec61g,EgfrOff03 3 NGG chr2 30122383 30122405 - exonic Phyhd1Off04 3 NAG chrX 13213424 13213446 - intronic Cask,Gpr34Off05 3 NAG chrX 11686178 11686200 - intronic BcorOff06 3 NAG chr16 7079230 7079252 - intronic Rbfox1Off07 3 NGG chr7 151526315 151526337 - intronic Shank2Off08 3 NGG chr9 49739346 49739368 - intergenic Ncam1, 2310014F07RikOff09 3 NAG chr1 58567252 58567274 + intergenic Orc2, Fam126bOff10 3 NGG chr10 109322284 109322306 + intronic Nav3Off11 3 NAG chr6 91915442 91915464 - intergenic 4930590J08Rik, Fgd5Off12 3 NGG chr19 58049718 58049740 - intronic Atrnl1Off13 3 NAG chr15 83055133 83055155 + intergenic Cyb5r3, A4galtOff14 6 NGG chr11 73029624 73029646 - intronic Shpk Off15 6 NAG chr3 118690448 118690470 + intronic DpydOff16 6 NAG chr5 67173306 67173328 - intronic Limch1Off17 6 NAG chr8 37646560 37646582 - intronic Dlc1Off18 7 NGG chr14 85943851 85943873 + intergenic Pcdh17, Diap3Off19 4 NGG chr4 81475756 81475778 + intergenic Mpdz, NfibOff20 2+ 1bp del NAG chr1 9344457 9344457 - intergenic Sntg1, Rrs1Off21 2+ 1bp ins NAG chr2 26861506 26861506 + intronic Adamts13

*MM, Number of mismatches in the 20mer target sequence; Chr, chromosome; Start and End, begin and end of the coordinates of the sites (NCBI37/mm9 mouse assembly).

Supplementary Table 2 Off-target sequences of Rubicon-E5F2 target site




PrimerRubicon-E5F2-F TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGCTCAGATCGATGCATCCARubicon-E5F2-R GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTGGATGCATCGATCTGAGCCT7-Rubicon-E5F2 TTAATACGACTCACTATAGGCTCAGATCGATGCATCCA

gRNA-R AAAAGCACCGACTCGGTGCCRubicon-F1 AGCAGAGGGTTTTATGCGCTRubicon-R1 CCACCACACCCAACTCTTCA

Rubicon-Off01-F2 GTTGCCTCACTCAGGATGGTRubicon-Off01-R2 CTCCTTTCCTGTCAGGCTGGRubicon-Off02-F1 GCTCCCCAGTGACCATGTAGRubicon-Off02-R1 GGATGAAGTATGGGCAGCCTRubicon-Off03-F1 TCCACCCCCTGTATGGTAGGRubicon-Off03-R1 ACTTGAGGGATCAAGCCAGCRubicon-Off04-F1 GGAAGAGGAAGTGGCTGGTGRubicon-Off04-R1 ACCTGGTACACAGAGAGGCARubicon-Off05-F1 GGGTGGCCAACCTTGAACTRubicon-Off05-R1 AAACCTGGTCCCAGGAGCTARubicon-Off06-F1 CCAGGAAGGACTGAGCATGGRubicon-Off06-R1 TCCGAGGCACTTGTAGATGCRubicon-Off07-F1 GGCTGCCTGACTCATGGAATRubicon-Off07-R1 CCAGACCTCCTCTGTCCTGARubicon-Off08-F1 ACTGGGTGCTAAATCAGGGCRubicon-Off08-R1 CTGAGAAAGCTACGACGGGGRubicon-Off09-F1 GGCCTCACCCAAAGGCTATTRubicon-Off09-R1 TCACACCAGCTGAACCAGTCRubicon-Off10-F2 TTCCACCAGCAGCATCTGAGRubicon-Off10-R2 TCTTGATGCCTGCAACCAAACRubicon-Off11-F1 ACCAGCAAGTACTCTTCGGCRubicon-Off11-R1 TGATAGGTTGTGAGGCGCAGRubicon-Off12-F1 TCAGTTGTGACTGGGGTTTAGGRubicon-Off12-R1 ACCTTCCTAGGGTGCTCACARubicon-Off13-F1 CATGAAAGCGGCCAGTGATGRubicon-Off13-R1 CTCAGCACCTGGGGGTAATCRubicon-Off14-F1 ACATGGAGGGTTCTGGGAGTRubicon-Off14-R1 ACCCCTTAGTTCATGCCAGCRubicon-Off15-F1 AAAAGACTTAGAGGCGCCCARubicon-Off15-R1 ACAATGGTAGCTCCGGATGACRubicon-Off16-F1 CTGCCCAGTGTGGGAGTTAGRubicon-Off16-R1 ACCCCAGAGTATCAGGGCATRubicon-Off17-F1 GGGATGCAGCGGATAGTCAARubicon-Off17-R1 CTCATCTGTGCCTGGCTGAARubicon-Off18-F1 TCTTCCTCTTCAGTTGCCTACARubicon-Off18-R1 GCTAAGGGTGCAGTTGTTGCRubicon-Off19-F1 GTCCAACTTTGCAAGGCCAGRubicon-Off19-R1 CCATGCCCCCTTTCCTGATTRubicon-Off20-F1 TAGCCCATCTCCTGCTCACTRubicon-Off20-R1 TGTACTGTGCCACCAGACAGRubicon-Off21-F1 TGTCTTTCATTGGCCCGGAGRubicon-Off21-R1 ACTGGAGTTGTGTGCCAGAG

Sequence

Supplementary Table 3 Sequencing and targeting primers for Rubicon-E2F2 target site




PrimerRubicon-E5F2-F TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGCTCAGATCGATGCATCCARubicon-E5F2-R GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTGGATGCATCGATCTGAGCCT7-Rubicon-E5F2 TTAATACGACTCACTATAGGCTCAGATCGATGCATCCA

gRNA-R AAAAGCACCGACTCGGTGCCRubicon-F1 AGCAGAGGGTTTTATGCGCTRubicon-R1 CCACCACACCCAACTCTTCA

Rubicon-Off01-F2 GTTGCCTCACTCAGGATGGTRubicon-Off01-R2 CTCCTTTCCTGTCAGGCTGGRubicon-Off02-F1 GCTCCCCAGTGACCATGTAGRubicon-Off02-R1 GGATGAAGTATGGGCAGCCTRubicon-Off03-F1 TCCACCCCCTGTATGGTAGGRubicon-Off03-R1 ACTTGAGGGATCAAGCCAGCRubicon-Off04-F1 GGAAGAGGAAGTGGCTGGTGRubicon-Off04-R1 ACCTGGTACACAGAGAGGCARubicon-Off05-F1 GGGTGGCCAACCTTGAACTRubicon-Off05-R1 AAACCTGGTCCCAGGAGCTARubicon-Off06-F1 CCAGGAAGGACTGAGCATGGRubicon-Off06-R1 TCCGAGGCACTTGTAGATGCRubicon-Off07-F1 GGCTGCCTGACTCATGGAATRubicon-Off07-R1 CCAGACCTCCTCTGTCCTGARubicon-Off08-F1 ACTGGGTGCTAAATCAGGGCRubicon-Off08-R1 CTGAGAAAGCTACGACGGGGRubicon-Off09-F1 GGCCTCACCCAAAGGCTATTRubicon-Off09-R1 TCACACCAGCTGAACCAGTCRubicon-Off10-F2 TTCCACCAGCAGCATCTGAGRubicon-Off10-R2 TCTTGATGCCTGCAACCAAACRubicon-Off11-F1 ACCAGCAAGTACTCTTCGGCRubicon-Off11-R1 TGATAGGTTGTGAGGCGCAGRubicon-Off12-F1 TCAGTTGTGACTGGGGTTTAGGRubicon-Off12-R1 ACCTTCCTAGGGTGCTCACARubicon-Off13-F1 CATGAAAGCGGCCAGTGATGRubicon-Off13-R1 CTCAGCACCTGGGGGTAATCRubicon-Off14-F1 ACATGGAGGGTTCTGGGAGTRubicon-Off14-R1 ACCCCTTAGTTCATGCCAGCRubicon-Off15-F1 AAAAGACTTAGAGGCGCCCARubicon-Off15-R1 ACAATGGTAGCTCCGGATGACRubicon-Off16-F1 CTGCCCAGTGTGGGAGTTAGRubicon-Off16-R1 ACCCCAGAGTATCAGGGCATRubicon-Off17-F1 GGGATGCAGCGGATAGTCAARubicon-Off17-R1 CTCATCTGTGCCTGGCTGAARubicon-Off18-F1 TCTTCCTCTTCAGTTGCCTACARubicon-Off18-R1 GCTAAGGGTGCAGTTGTTGCRubicon-Off19-F1 GTCCAACTTTGCAAGGCCAGRubicon-Off19-R1 CCATGCCCCCTTTCCTGATTRubicon-Off20-F1 TAGCCCATCTCCTGCTCACTRubicon-Off20-R1 TGTACTGTGCCACCAGACAGRubicon-Off21-F1 TGTCTTTCATTGGCCCGGAGRubicon-Off21-R1 ACTGGAGTTGTGTGCCAGAG

Sequence

Primer SequenceRubicon N-terminus For. TCAGGGTGTAGTGCATGGTTRubicon N-terminus Rev. GACGCATCCGGACTATAAGGRubicon C-terminus For. GTCAGGCCACAAGCCTATAARubicon C-terminus Rev. AGCAGTAAGGAACCCACTATTC

mIL-1β For. AAGGAGAACCAAGCAACGACAAAAmIL-1β Rev. TGGGGAACTCTGCAGACTCAAACTmIL-6 For. GACAACTTTGGCATTGTGGmIL-6 Rev. ATGCAGGGATGATGTTCTG

mIL-12p40 For. ACAGCACCAGCTTCTTCATCAGmIL-12p40 Rev. TCTTCAAAGGCTTCATCTGCAA

mTNFα For. ATGAACGCTACACACTGCATCmTNFα Rev. CCATCCTTTTGCCAGTTCCTCmActin For. ATGGAGGGGAATACAGCCCmActin Rev. TTCTTTGCAGCTCCTTCGTT

Supplementary Table 4 Real-time PCR primers


Documents

ULEMENTARY INORMATION - static-content.springer.com10.1038/ncb3192... · of the introduced stop codon (C terminus). Data are presented as mean ± SD (n=3 independent experiments,