Fig 2: Knock-out of GPRC5A results in impaired activation of FAK in adhering cells. (A) A representative Western blot demonstrating FAK phosphorylation dynamics in MDA-MB-231 cells at different time points after plating on Collagen I (0.1 mg/mL). (B) Quantification of the Western blot shown in (A) for 30 min after plating. Phosphorylated FAK signal was normalized to the loading control (GAPDH) and total FAK signal. Data represent the mean ± SEM, N = 3. Statistical significance was evaluated using one-way ANOVA test. *, p < 0 .05. (C) Representative Western blot demonstrating the amount of FAK co-immunoprecipitated with Src in control and GPRC5A knock-out MDA-MB-231 cells 30 minutes after plating on Collagen I (0.1 mg/mL). (D) Quantification of the Western blot shown in (C). Src-co-immunoprecipitated FAK signal was normalized to loading control and total FAK signal of the input samples. Data represent means ± SEM, N = 3. Statistical significance was evaluated using one-way ANOVA test. *, p < 0 .05.

Fig 3: NID1 promotes cancer cell migration, invasion, adhesion to ECs, and tube formation in vitro. (A,B) Transwell migration (A) and invasion (B) assays of MDA-MB-231 vector and NID1-overexpressing cells. (C,D) Transwell migration (C) and invasion (D) assays of LM1a control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2). (E,F) Attachment of vector or NID1-overexpressing MDA-MB-231 cells to HPMEC-ST1.6R EC monolayers. (F) Vector cells were pretreated with serum-free medium (SFM) or CM from vector or NID1-overexpressing MDA-MB-231 cells. (G) Western blot analysis of protein levels of focal adhesion kinase (FAK) and its phosphorylated form, pFAK Y397, in MDA-MB-231 vector control and NID1-overexpressing cells as well as ECs before and after incubation with cancer cells. Quantification of relative amounts of phosphorylated FAK (pFAK/FAK) in ECs is visualized; expression was normalized to the no cell control. (H) Western blot analysis of protein expression of integrin subunits α3, αv, β1, and β3 in ECs and HPL1 after incubation with CM from vector or NID1-overexpressing HTB140 cells. Expression of β-actin was used as a loading control. (I) Attachment of HTB140 cells to HPMEC-ST1.6R EC monolayers after pretreatment with CM from vector control and NID1-overexpressing HTB140 cells followed by treatment with various integrin-blocking antibodies, control IgG, or SFM. (J,K) Immunofluorescence images (J) and quantification (K) of vascular permeability by the area occupied by diffused dextran (K, left) and the number of leaked sites (K, right) after treatment of mice with CM derived from vector and NID1-overexpressing MDA-MB-231 cells. n = 10 mice. (L) Quantification and phase images of in vitro tube formation of MDA-MB-231 vector control and NID1 overexpression cells. Bar, 100 µm. In vitro assays were performed at least in triplicate. All data represent the mean ± SEM. (n.s.) Not significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001, Student's t-test.

Fig 4: CASC9 activates FAK-PI3K/Akt signaling pathways through LAMC2. a Western blot analysis showed altered levels of pFAK, pPI3K, and pAkt after CASC9 or LAMC2 knockdown in KYSE150 and KYSE450 cells. b Western blot analysis of FAK-PI3K/Akt signaling pathways of the rescue experiment. c RT-qPCR analysis of MMP10 and MMP13 expression. d, e The expression of MMP10 and MMP13 in KYSE150 and KYSE450 cells after CASC9 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: TET1 activates PTEN transcription mediated by its catalytic domainA. qPCR analysis of TET1 expression level in KD3 (upper panel) and SGC-7901 (lower panel) cells after over-expression of wild type or mutant TET1. B. Over-expression of mutant TET1 has no effect on PTEN, p53, hMLH1 and IRX1. C. Over-expression of wild type but not mutant TET1 increases 5-hmC and PTEN level, decreases p-AKT and p-FAK in KD3 cell. D. Over-expression of wild type but not mutant TET1 increases PTEN level, decreases p-AKT and p-FAK in SGC-7901 cell. E & F. Over-expression of wild type but not mutant TET1 inhibits cell migration and invasion. * p<0.05; ** p<0.01.