Extended Data Fig. 2: TFPI2 regulates GSC self-renewal, proliferation, and apoptosis.

a, b, Representative of tumorspheres of QPP7 (a) and CT2A (b) cells expressing control shRNA (shC) and Tfpi2 shRNAs (shTfpi2). Scale bar, 200 µm. c, d, Representative of proliferation in shC and shTfpi2 QPP7 (c) and CT2A (d) cells. e–j, Representative and quantification of flow cytometry for apoptosis in GSC272 (e, f), QPP7 GSCs (g, h) and CT2A cells (i, j) expressing shC and shTFPI2. k, Immunoblots for cleaved caspase 3 (CC3) in cell lysates from GSC272, QPP7, and CT2A cells expressing shC and shTFPI2. l, Immunoblots for CD133 and SOX2 in cell lysates from QPP7 GSCs expressing shTfpi2 with or without reexpression of shRNA-resistant TFPI2 cDNA (OE). m, Representative of tumorspheres in QPP7 GSCs expressing shTfpi2 with or without TFPI2 OE. Scale bar, 400 µm. n, Immunoblots for TFPI2 in cell lysates from TFPI2 wild-type (WT) and different clones of CRISPR knockout (KO) GSC272. o, Immunoblots for CD133 and SOX2 in cell lysates from TFPI2-WT and TFPI2-GSC272. p, Representative of tumorspheres in TFPI2-WT and TFPI2-KO GSC272. Scale bar, 400 µm. q, r, Representative (q) and quantification (r) of immunofluorescence staining of SOX2 in size-matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 50 µm. s, t, Representative (s) and quantification (t) of immunochemistry staining of CD133 in tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 200 µm. u–x, Representative and quantification of immunofluorescence staining of Ki67 (u,v) and CC3 (w,x) in tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 50 µm. In (f, h, j, r, t, v, x), n = 3 biological replicates. Error bars indicate mean ± SEM. One-way ANOVA test.