Fig 2: Silencing HDGF in RLW ABs attenuated profibrotic genes in LX2 cells. RT-PCR analysis of (a) COL1A1 mRNA, (b) TIMP1 mRNA, and (c) TGFβ mRNA. (d) MMP2 mRNA. Data are from 3 independent duplicate experiments presented as mean ± SEM. Bars with * are significantly different from each other (p ≤ 0.05).

Fig 3: Erdafitinib and quisinostat are synergistic in BC cells with FGFR3 activating mutations.a Schematic diagram of FGFR3 S249 mutation. Red arrowhead represents the position of S249C in FGFR3. b MacSynergy II calculation (95% confidence interval) of the synergy between erdafitinib and quisinostat in UM-UC-14 cells, which is a BC cell line with FGFR3 S249C mutation. Cells were treated by different concentrations of erdafitinib and/or quisinostat for 3 days. And cell viabilities were determined by WST-1 assay and normalized to DMSO control. Synergistic inhibition was calculated by MacSynergy II. Synergistic inhibition above 0 means synergy, equal to 0 indicates additivity, and below 0 suggests antagonism. c Cell viability of UM-UC-14 cells by the treatment of erdafitinib and/or quisinostat. Cells were treated by erdafitinib and/or quisinostat for 3 days. And cell viabilities were determined by WST-1 assay and normalized to DMSO control. Data were plotted as mean ± standard deviation from three biological replicates and statistics were calculated by one-way ANOVA (****p < 0.0001). 2.5E, 2.5 nM erdafitinib; 2.5Q, 2.5 nM quisisnostat. d Western blots showing the FGFR3 and HDGF expression level with or without quisinostat treatment in UM-UC-14 cells. Cells were treated by quisisnostat for 2 days and then harvested for western blotting. β-actin (ACTB) was used as standard loading control. 10/25/50Q, 10/25/50 nM quisisnostat.

Fig 4: Quisinostat can also sensitize BC cells to erdafitinib by downregulating HDGF.a RNA-seq results showing the representative genes that are downregulated by quisinostat in all three BC cells. b RT-qPCR results showing the HDGF mRNA level with or without quisinostat treatment. Cells were treated by quisisnostat for 2 days and then harvested for RT-qPCR. All results were first normalized to β-actin (ACTB) loading control and then normalized to DMSO control of each cell line. Data were plotted as mean ± SEM from four biological replicates and statistics were calculated by t tests (****p < 0.0001). c Western blots showing the HDGF protein expression level with or without quisinostat treatment. Cells were treated by quisisnostat for 2 days and then harvested for western blotting. β-actin (ACTB) was used as standard loading control. d Quantification of (c) by ImageJ. Results were normalized to DMSO control for each cell line. Data were plotted as mean ± standard deviation from three biological replicates and statistics were calculated by t tests (****p < 0.0001). e TCGA analysis of the bladder cancer cohort showing that HDGF expression is higher in bladder cancer patients than in healthy tissue. Statistics were calculated by one-way ANOVA (*p < 0.05). Center lines represent the median value, upper and lower bounds of the boxes denote the upper and lower quartiles, upper and lower whiskers represent the 1.5x interquartile range, and data points outside the upper and lower whiskers are considered outliers. f Western blots showing that both siRNAs of HDGF (siHDGFs) can successfully knock down HDGF without affecting the expression of FGFR3 fusions in SW780 and RT112 cells. β-actin (ACTB) was used as standard loading control. C, siRNA of non-targeting control. g, h Cell viabilities of SW780 cells (g) and RT112 cells (h) treated by erdafitinib with or without HDGF knockdown. Cells were treated by erdafitinib for 3 days. And cell viabilities were determined by WST-1 assay and normalized to DMSO control for each cell line. Data were plotted as mean ± standard deviation from three biological replicates and statistics were calculated by one-way ANOVA (**p < 0.01; ****p < 0.0001). siControl, siRNA of non-targeting control. 10/25/50E, 10/25/50 nM erdafitinib; 50Q, 50 nM quisinostat.

Fig 5: Functional validation of HDGF in OS in vitro and in vivo. (A) Western blot. (B) CCK-8 assays. (C) Colony formation assay. (D) Transwell migration and invasion assays. (E) xenograft tumors derived from control and HDGF-knockdown cells. (F) Tumor growth curves. (G) Tumor weights in the control and HDGF-knockdown groups. (H) H&E, HDGF, and Ki67 staining of xenograft tumor sections. **P < 0.01, ***P < 0.001.