Fig 2: CD109 silencing modifies glioblastoma stroma.(A) Representative micrographs of BT12 xenografts immunostained with anti–collagen IV (green) and anti-podocalyxin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 µm. Arrows indicate empty basement membrane sleeves devoid of endothelium. (B) Quantification of collagen IV+/PODXL– empty sleeves in control and CD109-silenced BT12 xenografts (n = 10). (C) Representative micrographs of BT12 xenografts immunostained with anti-human vimentin (green) and anti-mouse CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 µm as indicated. Quantification of the average tumor blood vessel size (D) and density (E) in control and CD109-silenced BT12 xenografts (n = 10). (F) Quantification of the secreted ANG1 and ANG2 levels from the cell culture supernatants of CD109-silenced and nontargeted control BT12 GSCs (n = 2). (G) Expression of ANGPT1 and ANGPT2 mRNA levels determined by total RNA-Seq in CD109-silenced and nontargeted BT12 GSCs at the indicated time points. (H) Representative micrographs of BT12 xenografts immunostained with anti-human NUMA (green) and anti-NG2 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 µm as indicated. (I) Representative micrographs of BT12 xenografts immunostained with anti–a-SMA (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 µm. (J) Quantification of the a-SMA+ tumor pericytes in control and CD109-silenced BT12 xenografts (n = 10). (K) Representative micrographs of BT12 xenografts immunostained with anti-NG2 (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 µm. (L) Quantification of the pericyte coverage in control and CD109-silenced BT12 xenografts (n = 10). P values were calculated by using the unpaired 2-tailed t test except for F and G, where a t test with Mann-Whitney post hoc test was used.

Fig 3: Human epiblasts are not constricted horizontally upon implantation(A) Schematic drawing of a human blastocyst upon implantation at embryonic day (D)7. Implantation into the maternal endometrium (beige) mediated by the polar TE (blue), overlying the epiblast/inner cell mass (magenta-purple). The implantation results in differentiation and invasion of the trophectoderm/ trophoblast, which is hypothesized to exhibit stretching and pulling forces on the epiblast (red arrows). After implantation, the epiblast acquires a bilaminar disc-like structure forming a flat oval (D8–9).(B) Analysis of epiblast shapes of embryos from the Carnegie Collection. EPIBLAST is indicated with a red dashed line. Blastocyst: Carnegie embryo 8663; D7.5: Carnegie embryo 8020; D8: Carnegie embryo 8155; D9: Carnegie embryo 8004; D11–12: Carnegie embryo 7700.(C) Quantitative analysis of epiblast circularity of Carnegie embryos from blastocyst to D11–12. Scatterplot and mean.(D) Quantitative analysis of epiblast aspect ratio (height versus width) of Carnegie embryos from blastocyst stage to D11–12. Scatterplot and mean.(E) IF staining of in vitro cultured embryos from D6 to D9. Red indicates OCT4 (D6–8) and OCT4 + PODXL (D9); blue indicates DAPI (D6–7 and D9) and AP2G (D8). Scale bars, 20 µm.(F) Quantitative analysis of the circularity of in vitro cultured embryos from D6 to D9. The circularity continuously decreases as the epiblast becomes more oval shaped. Scatterplot, mean ± SEM. Analysis, unpaired Student’s t test: D6–D9, p = 0.0042. D6, n = 7; D7, n = 10; D8, n = 20; D9, n = 21.(G) Quantitative analysis of epiblast aspect ratio (height/width) of in vitro cultured embryos from (F). Aspect ratio decreases significantly upon implantation. D7–D9, p = 0.0219.(H) Y27632 treatment of mouse embryos in attachment culture for 20 h. Treated embryos were cultured in 100 µM Y27632, and controls were cultured in DMSO. Embryos stained for Oct4 (green), Gata6 (blue), pMyosin-II (red), and DAPI (gray). Experiment was carried out 3 times. Dashed white line encircles epiblast lineage.(I) Analysis of epiblast coverage angle by the primitive endoderm in Y27632-treated embryos versus controls. The controls are significantly more highly covered than the treated embryos, in which the coverage angle in several cases inverted with epiblast spreading over the primitive endoderm instead. Analysis, unpaired Student’s t test: p = 0.0425. Controls, n = 10; treated, n = 9.

Fig 4: Characterization of TRPC6 expression and GSK1702934A response in murine podocytes.A, platelet and glomerular lysates from wild-type (WT) and Trpc6-/- (KO) female animals were analyzed by SDS-PAGE and Western blot. Anti-TRPC6 antibodies (top) detected a major band around 105 kD in WT, but not KO, platelets. No corresponding specific band is seen in glomerular lysates. A faint band of a size similar to TRPC6 is seen in both WT and KO glomerular samples, suggesting a non-specific signal. Podocalyxin (Podxl, middle) and Erk1/2 (bottom) blots are shown as loading controls. B, time-course of Fura-2 fluorescence ratio (340/380) in primary podocytes cultured from Trpc6E896K/E896K (KI) glomeruli. After 30 seconds (dashed vertical line), cells were stimulated with vehicle (Veh), GSK1702934A (GSK; 50 µM), ADP (100 µM), ATP (100 µM), thrombin (Thr; 0.5 u/ml), or thapsigargin (Thap; 1 µM). Shown are mean ± SD; n = 4 using podocytes from 2 different animals. Post-stimulation (C) peak Fura-2 fluorescence ratio, and (D) area under the curve (AUC, arbitrary units) were calculated. Shown are the mean and values from individual experiments. RM one-way ANOVA with Dunnett’s multiple comparisons test to vehicle control.

Fig 5: Treated CAG-tetOG4 ESCs Express Endodermal Markers and Contribute to Primitive Endoderm (PrEn) in Chimeras(A) Gata4 mRNA expression in CAG-tetOG4 ESCs in N2B27 2iLIF (left, n = 4), FC 2iLIF (center, n = 4), and IDG 2iLIF (right, n = 3) after 6 h Dox treatment or control. Error bars, SD.(B) Top panel: CAG-tetOG4 ESC aggregates in control condition (top row) or Dox (bottom row), analyzed after 24 h for Gata4 (green, Alexa488), Sox17 (red, Alexa647), mCherry (gray), and DAPI (blue) (control, 49/49 structures; Dox, 37/37 structures; n = 3 each). Scale bar, 20 µm. Bottom panel: same as top, analyzed for Gata6 (green, Alexa488), Oct4 (red, Alexa647), mCherry and Podxl (gray, Alexa568), and DAPI (blue) (control, 49/52 structures; Dox, 45/45 structures; n = 3 each). Scale bar, 20 µm control and 15 µm Dox. Endogenous CAG-GFP in the green channel of control but downregulated in Dox (see Figure S1A). Below, quantification of the percentage of cells with the specified marker combinations in control and Dox aggregates. In the graphs, each dot is an aggregate.(C) Schematic of chimera aggregation: CAG-tetOG4 ESCs treated with Dox for 6 h or untreated and aggregated with E2.5 wild-type embryos. Contribution to either EPI or PrEn was assessed at E4.5. TE, trophectoderm.(D) Chimeras as in (C) analyzed for Sox17 (gray), CAG-GFP (green, with aGFP), and DAPI (blue). Contribution to PrEn with Dox-treated cells (bottom rows): 17/43 embryos from 3 females, 39%. Contribution to EPI of control cells (top rows): 22/22 embryos from 3 females. Scale and zoomed scale: 20 µm. Arrows, Sox17/CAG-GFP+ve cells; arrowhead, Sox17+ve/CAG-GFP-ve cells. In the graph, percentage of inner cell mass cells with PrEn identity was quantified. Each dot is an embryo.(E) CAG-tetOG4 ESC aggregates generated by combining cells either grown in control (top row), or Dox-treated cells for 6 hr (middle) or a 50:50 mixture of control and Dox-treated cells (bottom) and analyzed after 72 h for Gata6 (green), Oct4 (red), Podxl (gray), and DAPI (blue). Control: 41/43 aggregates, Dox: 55/56, 50:50: 60/65; n = 3 each. Scale bar, 20 µm. *p < 0.05,**p < 0.01, ****p < 0.0001, ns, nonsignificant.See also Figure S1.