Fig 1: TGFBI functions via binding to integrin avß5 on GSCs. (A) Schematic diagram of TGFBI protein structure. It contained a secretory signal (SP) in the N-terminal cysteine-rich domain (CRD), followed by four fasciclin-1 domains (FAS1 1-4) and Arg-Gly-Asp (RGD) in the C-terminal. The RGD motif could bind the integrins. (B) GSEA of the TGFBI high expression versus low expression in TCGA GBM RNA-seq indicated the integrin-binding pathway was significantly upregulated in the TGFBI high expression group. (C) qRT-PCR analyses of the expression of the av, a3, a5, a6, ß1, ß3, and ß5 in GSCs (456 and 3691) cultured with rhTGFBI or control. N = 3, ***P < 0.001, ****P < 0.0001, ANOVA, analysis of variance. (D) IB analysis of ITGAV in GSCs (456 and 3691) cultured with rhTGFBI or control. (E) Representative immunofluorescence (IF) staining of TGFBI and the potential receptors (ITGAV and ITGB5) in human GBM were shown. Scale bar represents 20 µm. (F) Representative IF images of ITGAV and TGFBI in mouse models. Scale bar represents 200 µm. (G) Co-immunoprecipitation of ITGAV and ITGB5 with the TGFBI-specific antibody from 456 and 3691 GSC cell lysates. Immunoglobulin G (IgG) was used as a control antibody for IP.
Fig 2: TGFBI triggers apoptosis and restores cisplatin sensitivity in NPC.a Western blot was performed on the whole-cell lysates (WCLs) of C666-1, NP69, and NP460 cells. Anti-TGFBI (aTGFBI) was used to assess endogenous TGFBI expression and anti-ß-actin (aß-actin) as the loading control. b Caspase-3 activity assay on C666-1 cells transfected with either empty vector (CTL) or TGFBI expression vector (Myc-tagged; TGFBI-Myc) for 48 h. Media was replaced with MEM 6 h after transfection. c Caspase-3 activity after incubation with cisplatin (CDDP) for an additional 20 h. Anti-Myc (aMyc-TGFBI) and anti-ß-actin (aß-actin) immunoblots are shown as controls for specificity and loading. d Cell viability, using ATPlite, of C666-1 cells transfected with empty vector (CTL) or TGFBI-expression vector (TGFBI-Myc), incubated for 24 h, re-seeded in 96-well plates, and treated with cisplatin for 72 h in MEM. e Western blot of NP69, or C666-1 WCLs 48 h after transfection with empty vector (CTL) or TGFBI-expression vector (Myc-tagged; TGFBI-Myc). Media was replaced with MEM 6 h after transfection. Immunoblots were performed using anti-TGFBI (aTGFBI), anti-PTEN (atotal-PTEN), anti-phospho PTEN (S380; apPTEN (S380)), anti-phospho AKT (S473; apAKT (S473)), anti-pan AKT (apan-AKT), and anti-ß-actin (aß-actin) as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. f Kaplan–Meier plot of DRFS for NPC patients dichotomized on the basis of high (>median) vs. low (=median) TGFBI mRNA expression level (n = 20). g IHC quantification of non-relapsed vs. relapsed NPC patient samples using an anti-TGFBI polyclonal antibody. The Kaplan–Meier curve was generated based on high (=median) vs. low (TGFBI expressed (cytoplasmic and membrane staining of tumor cells) (n = 30)
Fig 3: Inhibition of osteoblast differentiation by Bo-786 CM was partially mediated through BIGH3. (A) Generation of Bo-786 RCC cells with knockdown of BIGH3. Knockdown of BIGH3 was confirmed by real-time PCR, Western blot assay, and ELISA. (B) Cell proliferation and cell viability of BIGH3 knockdown cell lines. (C) Effects of CM from BIGH3 knockdown cell lines on PMO differentiation. ALP mRNA (left), ALP staining (middle), and ALP activity (right). (D) Osteocalcin mRNA and protein levels. (E) Mineralization. “NS”: 786-shNS nonsilencing control cells; “#3”: 786-shBG#3 cells; “#4”: 786-shBG#4 cells. *P < .05; **P < .01; “a”: compared to “Con”; “b”: compared to “NS”.
Fig 4: Mass spectrometry analysis of Bo-786 CM. (A) SDS-PAGE analysis of Bo-786 CM. The proteins were stained with Coomassie blue. (B) Lists of 25 secretory proteins with highest protein score from tandem mass spectrometry analysis. (C) Expression of BIGH3 in kidney cell lines as determined by real-time PCR and Western blot. (D) SDS-PAGE analysis of BIGH3-his7 protein expressed and purified from HEK293 cells (left). Western blot of purified BIGH3-his7 protein and Bo-786 CM with anti-BIGH3 antibody (right).
Fig 5: TGFBI can de-differentiate differentiated GSCs. (A) The mRNA level of GFAP and SOX2 in three different culture systems 45. *p < 0.05, Student's t-test for SOX2, ANOVA, analysis of variance for GFAP. (B) IB analysis of GFAP, SOX2 in GSCs (456 and 3691) cultured in serum-induced differentiation medium over a 15-day span. (C, D, E) qRT-PCR analyses (C), IB analyses (D), Representative IF images (E) of the GSC markers (CD133), the differentiation markers (GFAP) in GSCs (456 and 3691) cultured with rhTGFBI or control. Scale bar represents 20 µm. N = 3, **P < 0.01, ***p < 0.001, Student's t-test. (F) The quantification of IF images (E) showed DGCs with rhTGFBI stimulation re-expressed of CD133 and decreased in GFAP content. N = 3, *p < 0.05, **P < 0.01, Student's t-test.
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