Figure 6.
TDP-43 regulates cholesterol metabolism by regulating SREBF2 and LDLR expression. (A and B) TDP-43 binds to the SREBF2 transcripts as well as the downstream genes controlled by SREBF2. (A) Schematic of CLIP. Human oligodendrocytic (M03.13) cell lysates were UV cross-linked, lysed, and then used for TDP-43 pull-down assay. (B) Potential binding targets such as SERBF2, HMGCR, HMGCS, LDLR, and positive control TARDBP transcripts were pulled down in the TDP-43 antiserum–treated samples. (C) Schematic of TDP-43 and SREBF2 RNAi experiments. (D) Acute knockdown of TDP-43 leads to down-regulation of (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Human oligodendrocyte cell lines (MO3.13) were transfected with control siRNA and siRNA against TDP-43 or SREBP2. Whole-cell lysates were probed with SREBP2, LDLR, and the loading control GAPDH. Quantifications of immunoblots for (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Quantifications were done with three independent batch of treatments (n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, #, P < 0.10; *, P < 0.05; **, P < 0.01. (E) Newly synthesized transcripts detection by RT-qPCR in the MO3.13 cells treated with nontargeted siRNA or siRNA against TDP-43, followed by the DRB treatment. qPCR using primer pairs against the first exon-intron junction for (i) SREBF2, (ii) LDLR, and (iii) RPLP0 detects the cDNAs from the cells collected in different intervals (0, 15, 30, and 60 min) after the removal of DRB. These gene expression levels were all normalized to no-treatment controls. Quantifications were done with three independent batch of treatments (n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, ****, P < 0.0001. (F) Global translation reduction in the MO3.13 cells with TDP-43 knockdown. The lysates from cells treated with puromycin (100 µg/ml) in 15-min intervals (0, 15, and 30 min) after the incubation nontargeted siRNA or siRNA against TDP-43 were applied onto the SUnSET assay. Quantifications were done with two independent batches of treatments with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate statistical significance, ****, P < 0.0001. (G) The MO3 cells were treated with either control or TDP-43 siRNA followed by puromycin incubation. The cells were lysed followed by immunoprecipitation with an anti-puromycin antibody, and then were blotted with LDLR antibody. Ctrl, control; N-term, N-terminal; WB, Western blot; MW, molecular weight.

TDP-43 regulates cholesterol metabolism by regulating SREBF2 and LDLR expression. (A and B) TDP-43 binds to the SREBF2 transcripts as well as the downstream genes controlled by SREBF2. (A) Schematic of CLIP. Human oligodendrocytic (M03.13) cell lysates were UV cross-linked, lysed, and then used for TDP-43 pull-down assay. (B) Potential binding targets such as SERBF2, HMGCR, HMGCS, LDLR, and positive control TARDBP transcripts were pulled down in the TDP-43 antiserum–treated samples. (C) Schematic of TDP-43 and SREBF2 RNAi experiments. (D) Acute knockdown of TDP-43 leads to down-regulation of (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Human oligodendrocyte cell lines (MO3.13) were transfected with control siRNA and siRNA against TDP-43 or SREBP2. Whole-cell lysates were probed with SREBP2, LDLR, and the loading control GAPDH. Quantifications of immunoblots for (i) full-length and (ii) N-terminal SREBP2 and (iii) LDLR protein levels. Quantifications were done with three independent batch of treatments (n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, #, P < 0.10; *, P < 0.05; **, P < 0.01. (E) Newly synthesized transcripts detection by RT-qPCR in the MO3.13 cells treated with nontargeted siRNA or siRNA against TDP-43, followed by the DRB treatment. qPCR using primer pairs against the first exon-intron junction for (i) SREBF2, (ii) LDLR, and (iii) RPLP0 detects the cDNAs from the cells collected in different intervals (0, 15, 30, and 60 min) after the removal of DRB. These gene expression levels were all normalized to no-treatment controls. Quantifications were done with three independent batch of treatments (n = 3) with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons tests was used to evaluate statistical significance, ****, P < 0.0001. (F) Global translation reduction in the MO3.13 cells with TDP-43 knockdown. The lysates from cells treated with puromycin (100 µg/ml) in 15-min intervals (0, 15, and 30 min) after the incubation nontargeted siRNA or siRNA against TDP-43 were applied onto the SUnSET assay. Quantifications were done with two independent batches of treatments with three biological replicates per experiment. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate statistical significance, ****, P < 0.0001. (G) The MO3 cells were treated with either control or TDP-43 siRNA followed by puromycin incubation. The cells were lysed followed by immunoprecipitation with an anti-puromycin antibody, and then were blotted with LDLR antibody. Ctrl, control; N-term, N-terminal; WB, Western blot; MW, molecular weight.

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