Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Validation of ex vivo BMRC/CTC using complementary imaging platforms. ( A ) Detection of ex vivo human CTCs by CellSearch CTC (epithelial) kit. The kit enumerates CTCs using DAPI, Pan-cytokeratin-PE (CK-PE), and CD45-APC staining. Upper panel shows a CTC clustering with a CD45+ cell (lymphocyte); lower panels show an individual CTC. Image resolution achieved in accordance with the USFDA-approved CellSearch platform. ( B ) Immunofluorescent (IF) staining of ex vivo BMRC using GCDFP15/MG cocktail. Image of a BMRC showing red IF staining against mouse primary antibody clones 23A3 (GCDFP-15) and 304-1A5 (MG), and green IF staining for rabbit clone 31A5 (MG). ( C ) IF images of single ex vivo epithelial and stem-like BMRCs and CTCs. ( D ) DEPArray images of ex vivo epithelial and stem-like BMRCs and CTCs. Image magnification: 10×. The DEPArray platform allows the capture, visualization, and downstream interrogation of single CTCs. Since the image resolution and magnification obtained from FDA-cleared CellSearch and DEPArray platforms cannot be altered by the user, we used IF to provide a high-resolution image of captured ex vivo BMRC/CTCs.

Article Snippet: IHC on mouse tissue using antibodies of mouse origin were performed using M.O.M. elite peroxidase kit; dual IHCs were performed using ImmPRESS Duet Double Staining HRP/AP Polymer Kit, and triple IHCs were performed by multiplexing with ImmPRESS-AP Anti-Rat IgG, Mouse Adsorbed Polymer Detection Kit (Vector Labs, Burlingame, CA, USA).

Techniques: Ex Vivo, Imaging, Staining, Clone Assay

Journal: Investigative ophthalmology & visual science

Article Title: Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

doi: 10.1167/iovs.02-1068

Figure Lengend Snippet: FIGURE 9. Immunohistochemistry of the retina and the ciliary body at 6, 24, and 48 hours after injection. Infiltrating OX42- and ED1-positive cells are green. The NPs fluoresced in red. OX42- or ED1-positive cells engulfing the NPs were costained and fluoresced in yellow.

Article Snippet: Sections were then incubated in humidified atmosphere at room temperature for 1 hour with the following specific antibodies at 1:50 (in PBS, 1% skimmed milk, 0.5% Triton): monoclonal mouse anti-rat ED1 staining myeloid cells such as macrophages (Serotec, Oxford, UK); monoclonal mouse anti-rat OX42/CD11b staining microglia, dendritic cells, granulocytes, and macrophages (Serotec); polyclonal rabbit anti-bovine glial fibrillary acidic protein (GFAP) staining astrocytes and activated Müller cells (DAKO, Glostrup, Denmark); and monoclonal mouse anti-rat anti-pan cytokeratin (CytoK) staining epithelial cells.

Techniques: Immunohistochemistry, Injection

Journal: PLoS ONE

Article Title: Mesenchymal Transition and PDGFRA Amplification/Mutation Are Key Distinct Oncogenic Events in Pediatric Diffuse Intrinsic Pontine Gliomas

doi: 10.1371/journal.pone.0030313

Figure Lengend Snippet: Panel A: Radial plot showing the expression of oligodendroglial markers in the two groups of DIPG, in log2 ratios related each other. The red circle represent the median expression level of the whole population of DIPG. Group 1 expresses higher levels of oligodendroglial markers than group 2 DIPG. Panel B: Morphological oligodendroglial differenciation in group 1 tumors (HES staining, ×40). Panel C: Morphological astrocytic differenciation in group 2 tumors (HES staining ×40). Panel D: Olig2 immunohistochemistry in a group 1 DIPG showing that probably not all cells in the biopsy are tumoral (×40). Panel E: Dual immunohistochemistry for Olig2 and GFAP showing that tumor cells in mitosis are GFAP negative but Olig2 positive (×100). Panel F: Overall survival of 55 DIPG according to the presence (red) or absence (blue) of oligodendroglial differenciation. Median OS was shorter in patients with oligodendroglial type of tumors (7.73 vs 12.37, p = 0.045, log rank test).

Article Snippet: Sections were then incubated with various commercial monoclonal primary antibodies against Olig2 (AF 2418, 1/150, R/D system, CA, USA), P53 (DO-1, 1/1, Ventana) and MIB-1 (1/100; Dako, Glostrup, Denmark).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a Kaplan–Meier curve of GBM model. 14 days after NSG mice were intracranially injected with 387 GSCs, they were treated with either vehicle ( n = 16, median survival 27 days) or LGK974 ( n = 16, median survival 28 days) 5 mg/kg twice a day for 14 days by oral gavage (Log-rank test p = 0.4973). b Immunoblot of TCF1 expression in GBM tissue of representative control or LGK974 treated animals in ( a ) (representative 2 samples from each group were shown out of 9 samples analyzed from each group). Sox2 and α-Tubulin were used as loading controls. c Multiple antigen immunohistochemistry staining of TCF1 (red, nucleus) and Sema3C (yellow, cytosol) in human GBM samples (representative pictures from 27 samples were shown). Left: 10x objective view with scale bar 200 μM. Right: 63x oil objective view with scale bar 50 μM. d Scatterplot and linear regression analysis of TCF1 and Sema3C positive stained cells in human GBM ( n = 27 GBM samples, simple linear regression test, R 2 = 0.66, p < 0.0001, Slope = 0.6034, 95% confidence interval 0.43 to 0.78). e Western blots of TCF1 and Sema3C after knockdown of TCF1, Sema3C, or both in 387 GSCs used in orthotopic xenograft model in figure panels ( f ) and ( g ). f Left panels: H&E staining of mouse brain panorama image. Scale bar 2 mm. Right panels: Ki67 immunohistochemistry staining in shNT, shSema3C, shTCF1, or shSema3C plus shTCF1 knockdown tumors. Scale bar 50 μM. Representative images of 387 GSC-derived xenograft tumor samples of euthanized animals (as in C) are shown (one sample from each group out of 14 was shown). g Kaplan–Meier curve of 387 GSC-derived orthotopic xenografts expressing shNT (median survival, 32 days), shSema3C (median survival, 103 days), shTCF1 (median survival, 143 days) or shSema3C + shTCF1 double knockdown (median survival not reached) ( n = 14 for each group). Log-Rank test, each group compared with control p < 0.0001; shSema3c vs. shTCF1, P = 0.7461; shSema3C vs. shSema3C + shTCF1, p = 0.0055; shTCF1 vs. shSema3C + shTCF1, p = 0.0035. Source data are provided as a Source data file.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Injection, Western Blot, Expressing, Control, Immunohistochemistry, Staining, Knockdown, Derivative Assay

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a , b Left panels: representative tumorsphere images of 387 ( a ) and 4121 ( b ) GSCs after control shNT, shSema3C#1, and shSema3C#2 knockdown. Scale bar 100 μM. Right panels: quantification of tumorspheres after Sema3C knockdown ( n = 4 biological replicates in each group, compared with control unadjusted p < 0.0001, error bars: S.D. Scale bar 100 μM). c , d In vitro extreme limiting dilution assay in Sema3C knockdown 387 GSCs ( c ) and 4121 GSCs ( d ). Tables show estimated stem cell frequencies in control shNT, shSema3C#1, and shSema3C#2 knockdown GSCs with 95% confidence intervals ( n = 24 replicates in each dose, compared with control unadjusted p < 0.0001, error bars: S.D.). e , f Left panels: representative immunofluorescence images of EdU incorporation assay in 387 ( e ) and 4121 ( f ) GSCs after control shNT, shSema3C#1, and shSema3C#2 knockdown. Right panels: quantification of EdU + cells over total cells (EdU in red, DAPI in blue, n = 3 independent experiments, 387 GSCs control vs. shSema3C#1 unadjusted p = 0.0002; control vs. shSema3C#2 unadjusted p = 0.0006. 4121 GSCs control vs. shSema3C#1 unadjusted p < 0.0001; control vs. shSema3C#2 unadjusted p = 0.0053, error bars: S.D. Scale bar 50 μM). g , h Cell viability of 387 and 4121 GSCs after Sema3C knockdown ( n = 6 biological replicates for each group, compared with control unadjusted p < 0.0001, error bars: S.D.). Source data are provided as a Source data file.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Control, Knockdown, In Vitro, Limiting Dilution Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a Western blots of TCF1, c-Myc, and c-Met proteins after Sema3C knockdown. Western blots were repeated at least twice. b qRT-PCR of TCF1 and C-myc in 3359 and 4121 GSCs after Sema3C knockdown ( n = 4 independent experiments, Mann Whitney U-test p < 0.03, error bars: S.D.). c qRT-PCR of Axin2 (3359 GSCs p < 0.0001; 4121 GSCs p = 0.013), CCND1 (3359 GSCs p = 0.0378; 4121 GSCs p = 0.045), c-Jun (3359 GSCs p = 0.0004; 4121 GSCs p = 0.035), C-myc (3359 GSCs p = 0.04; 4121 GSCs p = 0.0063) and TCF1(3359 GSCs p = 0.019; 4121 GSCs p = 0.0059) in GSCs expressing FLAG vector or FLAG-Sema3C ( n = 3 independent experiments, Two-tailed T-test, error bars: S.D.). Source data are provided as a Source data file.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, MANN-WHITNEY, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a Western blots of β-catenin in cytosolic and nuclear fractions (left) of GSCs after Sema3C knockdown (right). Western blots were repeated at least twice. b Quantification of nuclear β-catenin positive cells of GSCs expressing FLAG vector or FLAG-Sema3C ( n = 3 independent experiments in 387 GSCs Two-tailed T-test p = 0.0066, error bars: S.D.; n = 4 independent experiments in 4121 GSCs, Two-tailed T-test p = 0.0088, error bars: S.D.). c , d Left panels: representative immunofluorescence images of β-catenin (green) in 387 ( c ) and 4121 ( d ) GSCs after control shNT and shSema3C knockdown. Nuclei were counterstained with DAPI (blue). Right panels: quantification of nuclear β-catenin positive cell fraction ( n = 3 independent experiments, Two-tailed T-test, p = 0.0012 (387 GSCs); p < 0.0001 (4121 GSCs) error bars: S.D. Scale bar 50 μM). Source data are provided as a Source data file.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Western Blot, Knockdown, Expressing, Plasmid Preparation, Two Tailed Test, Immunofluorescence, Control

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a Western blots of TCF1 and c-Myc proteins after PlexinD1 knockdown in GSCs. b Western blots of Rac1-GTP and Rac1 after Sema3C knockdown in GSCs. c Western blots of TCF1 and c-Myc in NSC23766-treated GSCs. d Western blots of TCF1 after Rac1 knockdown in GSCs. e Western blots of TCF1 and c-Myc in GSCs expressing FLAG-Rac1-Q61L in the setting of Sema3C knockdown. Immunoblots using whole cell lysates (left) and cytosol and nuclear fractions (right) are shown. Source data are provided as a Source data file. Western blots were repeated at least twice.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Western Blot, Knockdown, Expressing

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a Western blots of TCF1 and c-Myc in GSCs treated with Porcupine inhibitor LGK974. Cells were treated with LGK974 or vehicle control for 24 h. b Western blots of TCF1 in LGK974 treated GSCs expressing FLAG vector or FLAG-Sema3C. Cells were treated with LGK974 100 μM or vehicle control for 24 h. Source data are provided as a Source data file. Western blots were repeated at least twice.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

doi: 10.1038/s41467-023-37397-w

Figure Lengend Snippet: a In vitro extreme limiting dilution assay in shSema3C, shTCF1 or both knockdowns. Tables below show estimated stem cell frequencies in control shNT, shSema3C, shTCF1, and shSema3C + shTCF1 knockdown GSCs ( n = 8 technical replicates in each dose, at least three biological replicates, ELDA test, comparing to shSema3C + shTCF1 knockdown GSCs, 387 GSCs: shNT p < 0.0001; shSema3C P = 0.0003; shTCF1 p = 0.0002; 4121 GSCs: shNT p < 0.0001; shSema3C p < 0.0001; shTCF1 p = 0.028). b Western blots of TCF1 and c-Myc in shSema3C knockdown GSCs treated with LGK974 100 μM or vehicle control for 24 h. Western blots were repeated at least twice. c Model of Sema3C regulation of the Wnt pathway in GSCs. Despite upstream Wnt pathway inhibition, Sema3C binds to the Neuropilin 1 (NRP1)—PlexinD1 receptor complex pathway to activate Rac1. Active Rac1 (Rac1-GTP) facilitates β-catenin nuclear translocation to drive Wnt target gene transcription. Source data are provided as a Source data file.

Article Snippet: Antibodies used are listed below: Sema3C (IB) (Thermo Fisher Scientific, Cat# PA5-24997); Sema3C (IHC) (R&D Systems, Cat#MAB1728); TCF1 (Cell Signaling Technology, Cat#2203); c-Myc (Cell Signaling Technology, Cat#5605); c-Met (Cell Signaling Technology, Cat#8198); β-catenin (BD Transduction Laboratories, Cat#610153); α-Tubulin (Sigma-Aldrich, Cat#T6199-200UL); FLAG (Sigma-Aldrich, Cat#F1804); H2B (Santa Cruz Biotechnology, Cat#sc10808); Rac1 (Cytoskeleton, Cat#ARC03) and Ki-67 (Novocastra, Cat#NCL-Ki67p).

Techniques: In Vitro, Limiting Dilution Assay, Control, Knockdown, Western Blot, Inhibition, Translocation Assay

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 6. Rescue of Tsc1fl/fl;Tyrp1-Cre mouse retinal phenotypes by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a). (A) Levels of senescence markers in P30 mouse retinas with indicated genotypes were analyzed by Western blot (WB). Relative amounts of proteins used in each sample were determined by WB detection of β-actin. (B) Distributions of mTORC1-active cells, which are positive to pS6, MG, which are positive to Sox9, and senescent cells, which are positive to β-gal and p53, were examined by immunostaining of the eye sections. Expression of those markers in the Cre-affected cells were determined by comparing the expression of the Cre reporter R26tdTom. (C) Numbers of Sox9-positive MG in the mouse retinas were counted and the relative numbers are shown in the graph. (D) R26tdTom-negative wild-type cells and R26tdTom-positive Cre-affected cells in the retinas were determined byFACS and shown in the graph. (E) The lengths of telomeres of the cells isolated from P30 mouse retinas with the indicated genotypes were compared with that of control sample, and their relative values are shown in the graph. Error bars denote SD. Numbers of samples analyzed are shown in the graphs. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Western Blot, Immunostaining, Expressing, Isolation, Control

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 7. Hif1a supports mTORC1-induced retinal progenitor cell (RPC) proliferation through the expression of glycolytic enzymes. (A) The Cre-affected cells in P0 mouse retinas with the indicated genotypes were visualized by R26tdTom Cre reporter. Distribution of mTORC1-active cells in the boxed areas was also examined by immunostaining of pS6 and shown in the second row. Proliferation and cell cycle progression of RPCs in the mouse retinas were also examined by 5-ethynyl-2′-deoxyuridine (EdU)/5-chloro-2'-deoxyuridine (CldU) labeling and chasing experiments as described in Materials and methods. (B) R26tdTom-positive Cre-affected cell population in the mouse retinas were quantified by FACS and shown in the graph. Error bars, SD. Numbers of samples analyzed are shown in the graph (four independent litters). (C) RPCs that had progressed from S-phase to G2/M-phase for 3 hr (EdU;pH3-positive); PRs that had been born for 12 hr (EdU;Otx2-positive); and RPCs that had reentered cell cycle for 15 hr (CldU;IdU-positive) in the mouse retinas were counted and shown in the graph. Error bars denote SD (n = 5, five independent litters). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Relative mRNA levels of Hif1a, hexokinase 2 (Hk2), and pyruvate kinase M2 (Pkm2) in P0 mouse retinas with the indicated genotypes were examined by real-time quantitative polymerase chain reaction (RT-qPCR). The y-axis values are relative 2-ΔCt values against those of Tsc1fl/+;Tyrp1- Cre mouse retinas. Error bars, SD (n = 4, three independent litters). (E) Expressions of the indicated proteins in the mouse retinas were examined by Western blot (WB). Relative amounts of the proteins used in each sample were determined by WB detection of β-actin. (F) Relative WB band intensities of the proteins are determined by the ImageJ software and shown in the graph. Error bars, SD (n = 3, three independent litters). (G) Distributions of Pkm2, Tom20, and hypoxyprobe-labeled proteins in the mouse retinas were examined by immunostaining. (H) P0 Tsc1fl/fl;Tyrp1-Cre littermate mice were injected with a chemical inhibitor of glycolysis (2DG) or mitochondrial oxidative phosphorylation (Metformin), and the eye sections of the mice were

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Expressing, Immunostaining, Labeling, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Injection, Phospho-proteomics