Fig. 9
CdCl2 treatment of Sertoli cells in vitro induces changes in the expression of multiple Maps. Sertoli cells treated with CdCl2 (3 µM) vs. control (Ctrl, 0.9% saline) for 24 h were used for bulk RNA-Seq, qPCR, and immunoblot (IB) analysis. For IF analysis, CdCl2 at 1 µM for 6 h incubation was used. A Heatmap analysis of Maps illustrating multiple genes of MAPs that were either up (red) or down (blue) regulated compared between control (Ctrl) and CdCl2 treated Sertoli cell groups. These data were from n = 6 independent experiments used for bulk RNA-Seq which have been deposited at NCBI as Sequence Read Archive data (see Materials and Methods) as reported [25]. B qPCR data showing results of different Maps of n = 3 experiments in which the control (Ctrl) was arbitrarily set at 1 against which statistical analysis of paired Student’s t-test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not significantly different. C Distribution of Map1a across Sertoli cells was examined by IF in which Map1a appeared as aggregates along the protofilaments of microtubules that stretched across the entire Sertoli cell cytoplasm, consistent with the reported function of Map1a by binding onto the microtubule protofilaments to promote MT stabilization. For -ve Ctrl (negative control), the primary antibody against Map1a was substituted by the corresponding IgG of the same animal species. Scale bar, 20 µm; 200 µm in inset. Results shown here are representative findings of a single experiment from n = 3 independent experiments which yielded similar results. D IB analysis showing negligible changes in the protein expression of Map1a, but relatively mild down-regulation of Kif15, after CdCl2 (3 µM) treatment for 24 h. These IB data are consistent with findings shown in Fig. 8 since notable changes in the expression of these proteins were detected 48 and 72 h after CdCl2 treatment. Interestingly, CdCl2 induced a considerable up-regulation of p-p38-MAPK but not total p38-MAPK. The bar graph in the lower panel are composite data of IBs shown in the upper panel with each bar representing a mean ± SD of n = 3 experiments. Each experiment has triplicate cultures. ***, P < 0.005 by paired Student's t-test; ns, non-significantly different