Fig 2: MAFG that released the transcriptional suppression from YAP promotes the stemness gene expression for tumor cell dedifferentiation. (A) MCF-7 cells were cultured on a rigid plate or in the 90-Pa fibrin gel for 48 h and then collected for RNA-seq. GO pathway enrichment analysis (P < 0.05) of upregulated differentially expressed genes (DEGs) in dedifferentiated MCF-7 cells. (B) Venn diagram of intersection of DEGs and transcription factor proteins in soft and stiff MCF-7 cells. The top 10 intersected genes were listed. The cutoff criteria of upregulated DEGs were log2 fold change > 2 and an adjusted P < 0.05. (C) Volcano plot of RNA-seq DEGs. The red dots and blue dots represent upregulated and downregulated DEGs based on a log-fold change of >1 and adjusted P < 0.05. ITGB8 and MAFG were shown as red dots. (D) The level of MAFG in B16 or MCF-7 cells cultured on a rigid plate or in the 90-Pa or 1,050-Pa 3D fibrin gel for 48 h was analyzed by Western blot. (E) Immunostaining of MAFG in MCF-7 cells cultured on a rigid plate or in the 90-Pa or 1,050-Pa 3D fibrin gel for 48 h. Scale bar, 10 μm. (F to H) ChIP-qPCR analysis was performed with an antibody to MAFG and SOX2 (F), NESTIN (G), or NANOG (H)-promoter-specific primers in MCF-7 cells cultured on a rigid plate or in the 90-Pa 3D fibrin gel for 4 days. (I) The NIH3T3 or HEK293T cells were co-transfected with SOX2 promoter-luciferase reporter PGL3 and pCMV-MAFG plasmid for 24 h followed by analysis of luciferase activity. (J) The expression of SOX2, NESTIN, or NANOG in SGCTRL or MAFG-SGs-MCF-7 cells cultured in the soft 3D fibrin gel for 48 h was analyzed by qPCR. (K) ChIP-qPCR analysis was performed with an antibody to YAP and MAFG-promoter-specific primers in MCF-7 cells cultured on a rigid plate or in the 90-Pa 3D fibrin gel for 4 days. (L) HEK293T cells were co-transfected with MAFG promoter-luciferase reporter PGL3 and pCMV-YAP plasmid for 24 h followed by analysis of luciferase activity. (M) The level of MAFG in SGCTRL, YAP-SG, YAP-SG/NLS-YAP, YAP-SG/NES-YAP, or YAP-SG/WT-YAP-B16 or MCF-7 cells cultured in the 90-Pa 3D fibrin gel was analyzed by Western blot. (N) The levels of ITGB8 in SGCTRL or YAP-SGs-B16 or MCF-7 cells cultured in the 90-Pa 3D fibrin gel were analyzed by Western blot. (O) Same as (K), except the ChIP-qPCR analysis was performed with ITGB8-promoter-specific primers. (P) Same as (L), except cells were transfected with ITGB8 promoter-luciferase reporter PGL3. (Q) The level of ITGB8 in SGCTRL, YAP-SG, YAP-SG/NLS-YAP, YAP-SG/NES-YAP, or YAP-SG/WT-YAP-B16 or MCF-7 cells cultured in the 90-Pa 3D fibrin gel was analyzed by Western blot. In (E) to (L), (O), and (P), n = 3 biological independent experiments. ***P < 0.001, by one-way ANOVA Bonferroni’s test (E to L, O, and P). The data represent mean ± SD.

Fig 3: RhoGDI recruited by integrin β8 mediates RhoA inactivation in a soft mechanical microenvironment. (A) The levels of kindlin 2 and talin in MCF-7 cells cultured in a flask or the 90-Pa or 1,050-Pa 3D fibrin gel for 48 h were analyzed by Western blot. (B) MCF-7 cells were cultured in a flask or the 90-Pa 3D fibrin gel for 48 h. Cell lysates were collected for immunoprecipitation with anti-ITGB8 or anti-IgG antibody. The expression of RhoGDI and ITGB8 was analyzed by Western blot. (C) The MCF-7 cells cultured in a flask or the 90-Pa fibrin gel were stained with anti-RhoGDI and ITGB8 antibodies and observed under ultrahigh super-resolution microscopy. Scale bar, 5 μm. (D and E) The levels of GTP-RhoA, total RhoA (D), p-YAP, or total YAP (E) in SGCTRL or RhoGDI-SGs-MCF-7 tumor cells were analyzed by Western blot. (F and G) Same as (D) and (E), except for the SGCTRL, RhoGDI-SG, or RhoGDI-SG/RhoGDI-OE MCF-7 tumor cells. (H) The levels of p-RhoGDI or total RhoGDI in MCF-7 cells cultured in a flask or the 90-Pa 3D fibrin gel for 48 h were analyzed by Western blot. (I and J) The levels of p-FAK, FAK (I), p-SRC, or SRC (J) in MCF-7 cells cultured in a flask or the 90-Pa 3D fibrin gel for 48 h were analyzed by Western blot. (K to M) The levels of p-RhoGDI (K), RhoGDI (K), GTP-RhoA (L), RhoA (L), p-YAP (M), or YAP (M) in MCF-7 cells treated with PBS, SRC inhibitor (Dasatinib, 5 μM), or FAK inhibitor (GSK2256098, 5 μM) for 48 h were analyzed by Western blot.

Fig 4: Schematic diagram of the ITGB8-YAP-MAFG pathway regulating the dedifferentiation in soft mechanical microenvironments. H-stiff tumor cells have high Src and FAK activities that result in strong YAP nuclear localization, where YAP binds to the MAFG gene promoter and heavily represses MAFG expression; M-stiff tumor cells have moderate Src and FAK activities and generate moderately strong YAP nuclear location to inhibit MAFG and β8 expression; in a soft mechanical environment such as 90-Pa fibrin gels, the low mechanotransduction may further weaken Src and FAK activities in M-stiff cells and relieve more repression of MAFG and β8; and as a positive feedback, the upregulated β8, upon sensing the mechanical force, binds more RhoGDI1 to inactivate YAP and promote tumor cell dedifferentiation.