Fig 1: A schematic model of CASC2 in normal cells and pancreatitis cells. In normal cells, the DNA methylation levels in the promoters of CtBPs are very low and there are no intracellular and environmental inflammation stimuli. Thus, the expression of CtBPs is maintained at a basal level. As a result, CtBPs proteins cannot efficiently associate with PCAF and c-MYC to assemble a complex to activate the expression of CASC2 and its targets IL6 and IL17. In pancreatitis cells, the decreased DNA methylation levels in the promoters of CtBPs and environmental inflammation stimuli activate the expression of CtBPs. The amplified CtBPs form a heterotetramer, which is then recruited by the c-MYC-PCAF complex to assemble the CPM transcriptional machinery. The activation of CPM complex specifically binds to the promoter CASC2 to induce its expression, further leading to the induction of IL6 and IL17.
Fig 2: c-MYC associated with PCAF and CtBPs to assemble a complex. (A) The Flag-c-MYC-associated complex. The pCDNA3-2×Flag (empty vector, EV) and pCDNA3-2×Flag-c-MYC plasmids were transfected into MIA PaCa-2 cells, respectively. The resulting cells were subjected to immunoprecipitation with the anti-Flag resin. The purified complexes were separated in an SDS-PAGE gel and incubated with a silver staining kit. The IgG and Flag-c-MYC were indicated by arrows. (B) c-MYC could pull down PCAF and CtBPs in vivo. Equal weight of pancreatic tissues from three AP patients was mixed and lysed, and 1/11 cell extracts were used as an input, and the other 10/11 cell extracts were equally divided into two parts, followed by immunoprecipitation with an IgG and anti-c-MYC antibody-associated protein A beads, respectively. The input and output proteins were used to determine protein levels of c-MYC, PCAF, CtBP1 and CtBP2, respectively. (C) c-MYC directly interacted with PCAF but not CtBPs in vitro. The MIA PaCa-2 cells were transfected with different plasmids including pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP2, pCDNA3-2×Flag + pCDNA3-6×Myc-PCAF, pCDNA3-2×Flag-c-MYC + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag-c-MYC + pCDNA3-6×Myc-CtBP2, and pCDNA3-2×Flag-c-MYC + pCDNA3-6×Myc-PCAF. The resulting cells were lysed and immunoprecipitated with an anti-Flag and anti-Myc resins, respectively, followed by immunoblots to examine the input and output proteins levels using anti-Flag and anti-Myc antibodies. (D) PCAF directly interacted with both c-MYC and CtBPs in vitro. The MIA PaCa-2 cells were transfected with different plasmids including pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP2, pCDNA3-2×Flag + pCDNA3-6×Myc-c-MYC, pCDNA3-2×Flag-PCAF + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag-PCAF + pCDNA3-6×Myc-CtBP2, and pCDNA3-2×Flag-PCAF + pCDNA3-6×Myc-c-MYC. The resulting cells were lysed and immunoprecipitated with an anti-Flag and anti-Myc resins, respectively, followed by immunoblots to examine the input and output protein levels using anti-Flag and anti-Myc antibodies. (E) CtBPs assembled a heterotetramer in vitro. The MIA PaCa-2 cells were transfected with different plasmids including pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag + pCDNA3-6×Myc-CtBP2, pCDNA3-2×Flag + pCDNA3-6×Myc-c-MYC, pCDNA3-2×Flag-CtBP1 + pCDNA3-6×Myc-CtBP1, pCDNA3-2×Flag-CtBP1 + pCDNA3-6×Myc-CtBP2, and pCDNA3-2×Flag-CtBP1 + pCDNA3-6×Myc-c-MYC. The resulting cells were lysed and immunoprecipitated with an anti-Flag and anti-Myc resins, respectively, followed by immunoblots to examine the input and output proteins levels using anti-Flag and anti-Myc antibodies.
Fig 3: Trx-1 interacts with G6PD and increases its activity. (A) Protein interactors of “Trx-1” were identified using the HuPortTM human protein chip, and the enrichment of related pathways was determined with reference to GO and KEGG pathways. (B) The HuPortTM human protein chip identified G6PD as an interactor of Trx-1. (C) Depiction of the binding of Trx-1 with G6PD as identified by the HuPortTM human protein chip. (D) The interaction between Trx-1 and G6PD was confirmed by co-IP assays. HEK293T cells transfected with Flag-G6PD and Myc-Trx-1 were immunoprecipitated with anti-Flag-tag or anti-Myc-tag antibodies, IgG was used as negative control. (E) SW480 cells were incubated under glucose starvation conditions for 24 h, and then cellular localization of Trx-1 and G6PD was determined by immunofluorescence staining. DAPI staining was performed to identify the nucleus. This fluorescence image was captured using a confocal microscope. The scale bar represents 25 µm. (F) Protein expressions of G6PD and Trx-1 determined by Western blotting in SW480 cells stably expressing GFP, Trx-1, shLuc or shTrx-1, after 24 h incubation in glucose-deprived medium. (G-H) G6PD activity in SW480 cells with Trx-1 overexpression or knockdown. (I-L) NADPH levels and NADP/NADPH ratio in SW480 cells with Trx-1 overexpression or knockdown. Data are shown as mean ± SEM; n = 3. **p < 0 01, and ***p < 0 001.
Fig 4: c-MYC specifically regulated the expression of CASC2. (A) The potential transcription factor binding sites on the promoter of CASC2. A 1500-bp length of the CASC2 promoter was predicted the transcription factor binding sites. One c-MYC, one SP1, one NF-?B and one c-JUN binding sites were found, and their positions were shown. (B) Knockdown or overexpression of c-MYC changed the expression of CASC2. Total RNA from Control-KD, c-MYC-KD1, c-MYC-KD2, Control-OE, and c-MYC-OE cells were applied to qRT-PCR analyses to measure the mRNA levels of c-MYC and CASC2. ***P<0.001. (C) Knockdown or overexpression of SP1 could not change the expression of CASC2. Total RNA from Control-KD, SP1-KD1, SP1-KD2, Control-OE, and SP1-OE cells were applied to qRT-PCR analyses to measure the mRNA levels of SP1 and CASC2. ***P<0.001. (D) Knockdown or overexpression of NF-?B subunits could not change the expression of CASC2. Total RNA from Control-KD, p50-KD1, p50-KD2, p65-KD1, p65-KD2, Control-OE, p50-OE and p65-OE cells were applied to qRT-PCR analyses to measure the mRNA levels of p50, p65 and CASC2. ***P<0.001. (E) Knockdown or overexpression of c-JUN could not change the expression of CASC2. Total RNA from Control-KD, c-JUN-KD1, c-JUN-KD2, Control-OE, and c-JUN-OE cells were applied to qRT-PCR analyses to measure the mRNA levels of c-JUN and CASC2. ***P<0.001.
Fig 5: TDP-43 Entry into Mitochondria Requires AGK Independent of Its Lipid Kinase Function(A) OMX-SR microscopy reveals that import of TDP-43 (Myc-tagged, red) into mitochondria (TIM44, blue) and TDP-43-induced relocation of DNA (anti-DNA, green) into the cytoplasm are ablated in HEK293T cells lacking the TIM22 regulatory subunit AGK (scale bars, 0.5 µm). Overview images are maximum-intensity projections (top) or 3D surface reconstructions using Imaris software (bottom). See also Video S2.(B and C) Spatial quantification by Imaris software for (B) the percentage of Myc-TDP-43 in mitochondria (TIM44) and (C) the percentage of DNA outside of mitochondria in control, AGK-/-, AGK-/- +WT, or AGK-/- +G126E HEK293T cells; 30 cells per group.(D) TDP-43-induced (WT, mutant A315T and Q331K) mtDNA release (cytosolic/total lysis, percent) is ablated in cells that lack AGK.(E) Treatment with the TDP-43 inhibitor peptide (PM1; 1 µM for 24 h) prevents induction of IFNB1 and TNF in TDP-43-ALS patient iPSC-MNs.Data are mean ± SEM from 3 independent experiments. The p values were calculated using one-way or two-way ANOVA to Ctrl in (B)–(D) or unpaired t test between healthy Ctrl and ALS patient iPSC-MNs in (E). *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S3.
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