Figure 4.

Spiropyrazoline oxindole 1a decreases stemness and increases differentiation of NSCs. Mouse NSCs were grown in differentiation medium, incubated with 12.5 or 25 μM 1a or DMSO (control) for 24 h and collected for Western blot and immunocytochemistry as described in Experimental. (A) Representative immunoblots (top) and corresponding densitometry analysis (bottom) showing decreased Sox2 (left) and increased βIII-tubulin (right) protein levels after 1a treatment. Ponceau staining was used as loading control. Data represent mean ± SEM of at least three independent experiments. ^*p < 0.05 and ^**p < 0.005 from respective control by Student's t-test. (B) Representative images of immunofluorescence detection of Sox2 (left) and βIII-tubulin (right) after 24 h of 1a treatment at 12.5 μM. Nuclei were stained with Hoechst 33258. Scale bar, 25 μm. (C) Flow cytometry histograms showing surface marker expression of stemness-associated CD133, after treatment with vehicle (gray) or 1a at 12.5 μM (red) for 24 h, and respective quantification of CD133-positive cells mean fluorescence intensity.