Fig 2: SSd exhibited cytotoxicity in primary hippocampal NPCs through NRAGE/NADE/NRIF apoptotic signaling. A, The procedure of the cultured primary hippocampal NPCs. B and C, Effect of SSd on cell proliferation and quantitative analysis of the levels of EdU+ cells. The NPCs were treated with SSd for 24 hours, and cell proliferation was measured using the EdU kit. Scale bar 100 μm. D, Effect of SSd on apoptosis of NPCs. The NPCs were treated with SSd for 24 hours, and cell apoptosis was detected using Hoechst 33342 staining. Scale bar 25 μm. E, Western blot analysis of cleaved caspase‐3 and quantitative analysis of the levels of cleaved caspase‐3. The neurospheres were treated with SSd for 24 hours and harvested for western blot analysis. F, Effect of SSd on cell differentiation of NPCs. The NPCs were incubated with the medium for differentiation and treated with SSd for 7 days followed by immunofluorescence analysis for MAP2 (red) and GFAP (green). Scale bar 50 μm. G, Effect of SSd on NRAGE/NRIF/NADE signaling in NPCs and quantitative analysis of the levels of NRAGE, NRIF, and NADE. Data are expressed as mean ± SEM. The results are representative of three independent experiments. # P < .05 versus the control. ## P < .01 versus the control. Abbreviation: NS, not significant versus the control

Fig 3: SSd regulated NRAGE/NRIF/NADE signaling in NPCs by inducing activation of p75NTR ICD. A, Western blot analysis of the effect of SSd on p75NTR activation in NPCs and quantitative analysis of the levels of p75NTR ICD. B, Effect of p75NTR ICD shRNA on the inhibition of p75NTR ICD expression and quantitative analysis of the levels of p75NTR ICD. The neurospheres were incubated with p75NTR ICD shRNA or sh‐NC for 12 hours, then cultured with the normal medium for 72 hours, followed by western blot analysis. C, Effect of p75NTR ICD shRNA on SSd‐activated p75NTR and quantitative analysis of the levels of p75NTR ICD. D, Effect of p75NTR ICD shRNA on SSd‐induced NRAGE/NADE/NRIF signaling and quantitative analysis of the levels of NRAGE, NADE, and NRIF. E, Effect of p75NTR ICD shRNA on SSd‐induced apoptotic signaling and quantitative analysis of the levels of p‐JNK and cleaved caspase‐3. F, Effect of p75NTR ICD shRNA on SSd‐induced neurospheres growth. Scale bar 100 μm. The neurospheres were exposed to SSd for 24 hours after the p75NTR ICD shRNA or sh‐NC transfection, followed by western blot analysis and bright‐field analysis. G, Effect of p75NTR ICD shRNA on SSd‐inhibited cell proliferation in NPCs. The NPCs were exposed to SSd for 24 hours after the p75NTR ICD shRNA or sh‐NC transfection, and cell proliferation was detected by the EdU kit. Scale bar 50 μm. H, Effect of p75NTR ICD shRNA on SSd‐induced cell differentiation of NPCs. After transfected with p75NTR ICD shRNA or sh‐NC, the NPCs were incubated with differentiation medium for 7 days in the absence or presence of SSd followed by immunofluorescence analysis for MAP2 (red) and GFAP (green). Scale bar 50 μm. Data are expressed as mean ± SEM. The results are representative of three independent experiments. # P < .05 versus the control. ## P < .01 versus the control. ** P < .01 versus the indicated group