Fig. 4: Role of Tmsb10 in the regulation of ciliogenesis via suppression of the RAS/ERK pathway.
From: Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway

a E16.5 W-EGFP cells were treated with 0.1, 1.0, or 10.0 nM siT10 or 10.0 nM siCnt, then cultured in serum-free medium for 24 h. Thereafter, the cells were subjected to immunostaining for the cilial marker protein ARL13B (red). Nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI) (blue). The enclosed area is enlarged at the top right. Arrows indicate primary cilia. Scale bar = 10 µm. b Ciliated cells detected in the studies above were counted. The ratios of the ciliated cells to all cells (%) are shown. n = 5. *p < 0.001. c W-EGFP cells were treated with 0.1, 1.0, or 10.0 nM siT10 or 10.0 nM siCnt, then subjected to testis reconstruction assay. The EGFP-positive cells (indicated by the relative EGFP-positive area) in the reconstructed testes were analyzed quantitatively after incubation for 21 days. n = 3. p < 0.001. d W-EGFP cells were cultured and treated with 0.1, 1.0, or 10.0 nM siT10 or 10.0 nM siCnt. Expression of the Gli1 gene in the cells was examined. The data were standardized using Rn18s. n = 3. p < 0.001. e Interactions between TMSB10 and RAS were examined. Whole-cell lysates were prepared from HEK293 cells overexpressing FLAG-TMSB10 or FLAG together with KRAS. Proteins interacting with TMSB10 were immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were subjected to immunoblotting using antibodies for pan-RAS and FLAG. The positions of molecular weight markers are indicated on the left. Full blot images are displayed in Supplemental Fig. 9. f Whole-cell extracts were prepared from E16.5 W-EGFP cells treated with siT10 or siCnt in the presence (+) or absence (−) of SAG. Levels of phospho-ERK (pERK) and ERK were examined by western blotting. g Signal intensities in the blots above were quantified as described in the Materials and Methods. The amounts of pERK relative to ERK are shown. n = 3. p < 0.01. h A schematic illustration summarizing the results so far. TMSB10 was assumed to suppress the RAS/ERK pathway by interacting with RAS. i W-EGFP cells were treated with siCnt (open bar), siT10 (light blue bar), siRas (dark blue bar), or both siT10 and siRas (red bar). These cells were then used for testis reconstruction. The reconstructed testes were cultured in the presence of SAG for 21 days. The EGFP-positive cells in the reconstructed testes were analyzed quantitatively. n = 3. p < 0.001. j W-EGFP cells were treated with siRNAs as above, and their effect on ciliogenesis was examined by immunostaining for ARL13B. Ciliated cells were counted and the ratios of these cells to all cells (%) are plotted. n = 5. *p < 0.001. Letters a, b, and c on the bars in c, d, g, and i denote significant differences between the cell groups.