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. 2022 Jan 31;12:827985. doi: 10.3389/fonc.2022.827985

Figure 3.

Figure 3

Characterization of the effects of vemurafenib on epithelial differentiation and proliferation in SEs. SEs from NHEK and HrasA5 cells were treated with vemurafenib (VEM) (1 and 5 µM) for up to 5 weeks and histology and immunostaining were performed at the indicated time points. (A) H&E staining of SEs from NHEK demonstrates accelerated cornification, particularly evident upon 5 µM VEM (left). Also, HrasA5 epithelia showed a time-dependent increase in cornification. In addition, invasion was seen after 3 (1 µM VEM) and 1 week (5 µM VEM), respectively (right). (B) Improved differentiation is confirmed by immunostaining for the early, KRT10, and the late differentiation markers FLG and KRT2 in the epithelium of the NHEKs (left) and the HrasA5 cells (right). The BM components COLVII (green), COLIV (red), and LAM (red) are expressed as contiguous lines in NHEK SEs at all time points and all conditions. Note that COLIV is expressed continuously and present throughout the DE; though enriched in the BM zone (left). In HrasA5 SEs, COLVII is generally reduced and lost at the invasive front. COLIV and LAM rather appear “bloated” with the tumor cells pushing through small gaps (right). (C) Same SEs stained for the proliferation marker H3S10ph (red), demonstrating very similar proliferation for all NHEK SEs (left). In HrasA5 SEs, proliferation is present throughout the epithelium (control). Under VEM, proliferation gets restricted to the basal compartment (right). All SEs are counterstained for the early differentiation marker transglutaminase 1 (TGM). (D) For quantification of proliferation SEs from NHEK and HrasA5 cells were costained with COLVII and KI67 and the number of proliferating cells (KI67+) correlated with BM (COLVII) length. Neither NHEKs nor HrasA5 cells showed a significant regulation in proliferation (n = 2, mean + SEM, one-way ANOVA + Dunnett’s multiple comparison test; ns, not significant). For all conditions, nuclei were counterstained with DAPI (blue). Treatment with the DMSO was used as control. Time specifications relate to time after starting the scale bar = 300 µm [for (A, C)]; scale bar = 150 µm [for (B)].