Fig 2: Combination of dasatinib and infigratinib overcomes intrinsic resistance. (A) Representative images of long term colony formation assay in the NIH-3T3 cell line panel upon treatment with a combination of dasatinib and infigratinib or DMSO control at the indicated doses for 2 weeks. (B) Bar plots showing the quantification of well coverage of the colony formation assay in panel A. Data for each cell line are normalised to DMSO control treatment (n = 3). Statistical analysis of drug treatment versus DMSO was performed by two-way ANOVA with Dunnett’s post-hoc multiple comparison adjusted p-value (*** p < 0.001). (C) Dose-response curves of the WT FGFR3, FGFR3-TACC3 and S249C cell lines upon treatment with infigratinib and dasatinib either as single agents or in combination for 72 h. For the combination arm, cells were treated with infigratinib and dasatinib in a 1:1 ratio with each drug at half the dose of the single agent. Cell viability is normalised to DMSO control treatment (n = 3). Statistical analysis of the combination arm versus either single agent at each dose was evaluated using two-way ANOVA with Dunnett’s post-hoc multiple comparison adjusted p-value at each drug dose (** p < 0.01, *** p < 0.001). (D) Immunoblot of Erk1/2 and Src phosphorylation levels in NIH-3T3 cells treated with a combination of infigratinib and dasatinib in a 1:1 ratio for 6 h, with the indicated concentration representing to total drug concentration used. Data presented for (B) and (C) represent mean ± SD. EV – empty vector control, WT – wildtype FGFR3, F3-TACC3 – FGFR3-TACC3, Inf – infigratinib, Das – dasatinib.

Fig 3: Enrichment analysis of downstream TFs and immunofluorescence of the INHBB-ACVR2A/B-pSMAD2 axes. (A) Enrichment analysis of the six top TFs in SSCs. (B) Ridge plot of the density distribution of FC of TGs for the six TFs. (C) Dual immunofluorescence of VASA (red) and INHBB (green) in adult human testicular paraffin sections. (D) Dual immunofluorescence of ACVR2B (red) and INHBB (green) in adult human testicular paraffin sections. (E) Dual immunofluorescence of ACVR2B (red) and pSMAD2 (green) in adult human testicular paraffin sections. (F) Dual immunofluorescence of FGFR3 (red) and ACVR1B (green) in adult human testicular paraffin sections. Triangles and circles indicate SSCs, and arrows indicate Sertoli cells. The scale bars represent 10 µm.

Fig 4: Developmental changes in the expression patterns of AQP4, FGFR3, and CD44. (A) At E96, AQP4 was expressed at LER and GER but not in hair cells and substantial sensory epithelium between inner hair cells and outer hair cells. (B) At E101, AQP4 expression was detected in some supporting cells. (C) At E115, AQP4 expression was detected in immature outer pillar cells (arrow), Hensen's cells, some GER cells, and outer sulcus cells. (D) At P0, AQP4 was expressed in inner sulcus cells, Hensen's cells, Claudius cells, and outer sulcus cells. (E) At E96, FGFR3 expression was detected in hair cells and some SOX2-positive cells. (F, G) At E101 and E115, FGFR3 expression was detected in outer hair cells and some SOX2-positive supporting cells. No expression was detected in inner hair cells. (H) At P0, FGFR3 expression was not detected. (I, J) No CD44 expression was detected at E96 and E101. (K) At E115, CD44 was expressed in both inner and outer pillar cells, and some Claudius cells and Hensen's cell. (L) At P0, CD44 expression was only detected in inner sulcus cells and SOX2-negative Hensen's cells. The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm. A, B, D, E, G, H, and I: basal turn; C, F, J, K, and L: middle turn. IHC: inner hair cells, OHC, outer hair cells; IPC, inner pillar cells; OPC, outer pillar cells; HC, Hensen's cells. E96 (n = 5), E101 (n = 4), E115 (n = 4), and P0 (n = 3).

Fig 5: FGFR3 staining is heterogeneous. (A) Representative images (50× magnification) of heterogeneously stained areas in 3 cases with strong FGFR3 staining. In sample GBM-05, the staining pattern clearly differed between tumor sites. Scale bar 50 µm. (B) Summary of FGFR3 IHC staining data from whole-mount tissue slides. Most samples (65% of all) retain their original FGFR3 staining score evaluated from TMA data, but especially moderate scoring was observed in many samples (21% of all) with negative to weak staining on TMA. Numbers of TMA samples, whole-mount tissue samples used for staining, IDH1 p.R132H staining-positive samples, and GBM samples are marked on the left-hand side. On the right, shown are the number of whole-mount tissue samples and their proportion of all the stained slides with the specified staining score on TMAs.