Figure 2: OSGIN1 is not transcriptionally regulated by P53 and does not induce cellular apoptosis in human spinal cord astrocytes.

(a) Schematic illustration of OSGIN1 under the transcriptional control of P53 in immortalized/transformed cells. P53 binds to the promoter region of OSGIN1 to induce transcription. Translated OSGIN1 protein physically interacts with P53 protein to induce cytochrome c release in mitochondria and subsequent apoptosis. (b–d) qRT-PCR of astrocyte RNA extracts. Data graphed as relative expression of gene modulation relative to control-siRNA (b,c) or control-siRNA with 0 μM MMF (d). Gene expression was normalized to 1 (dashed line). (b,c) qRT-PCR for P53 (b) and OSGIN1 (c) mRNA following transfection with control-, OSGIN1-, or P53-siRNA. Mean ± SEM shown for 4 independently performed experiments with n = 2 to 4/condition. ****p < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons) compared with control-siRNA. (d) qRT-PCR for OSGIN1 mRNA following transfection with control- or P53-siRNA. Mean ± SEM shown for 3 independently performed experiments with n = 2/condition. **p < 0.01 (one-way ANOVA with Tukey’s multiple comparisons) compared with control-siRNA. (e,f) Quantification of cellular apoptosis using TiterTACS™ in human spinal cord astrocytes. Data graphed as fold change relative to 0 μM MMF and normalized to 1 (dashed line). Nuclease-treated cells were included as a positive control (+Con). (e) Quantification of apoptosis following 0, 10, or 30 μM MMF. n = 4/condition. ****p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons) compared with 0 μM MMF. (f) Quantification of apoptosis following transfection with control-, NRF2-, OSGIN1-, or P53-siRNA and treated with 0, 10, or 30 μM MMF. n = 6/condition. *p < 0.05 and ****p < 0.0001 (two-way ANOVA with Tukey’s multiple comparisons) compared with control-siRNA treated with 0 μM MMF.