Journal: Signal Transduction and Targeted Therapy

Article Title: Short-chain acyl-CoA dehydrogenase initiates mtDNA demethylation and leakage to fuel antitumor immunity in colorectal cancer

doi: 10.1038/s41392-026-02675-8

Figure Lengend Snippet: ACADS deficiency promotes mitochondrial DNA methyltransferase 1 (mito-Dnmt1)-dependent mtDNA methylation and leakage suppression. a LC-MS/MS results showing the interaction of Acads and Dnmt1 in MC-38 cells. b Lysates from MC-38 cells prepared in IP lysis buffer were incubated for 8 h with magnetic beads conjugated to an anti-Acads antibody or an IgG control. After washing, the proteins bound to the beads were eluted with 2× loading buffer and detected via Western blotting with antibodies against Dnmt1 and Acads. c MC-38 cells grown on coverslips were divided into four groups. After fixation, permeabilization, and blocking, the cells were incubated with different primary antibodies (or controls): BSA (control, Con.), rabbit anti-Acads, mouse anti-Dnmt1, or a combination of both antibodies. Following washing, the samples were incubated with corresponding secondary antibodies conjugated to fluorescent probes. Signal amplification was then performed via a DNA polymerase-mediated method. Nuclei were counterstained with DAPI (blue), and images were acquired. The figure shows representative immunofluorescence images from the four treatment groups (scale bar, 20 μm). d Immunofluorescence staining was performed on the two indicated groups (control and the combination of rabbit anti-Acads with mouse anti-Dnmt1), followed by imaging via confocal microscopy (scale bar, 5 μm). Representative images of the negative control group are presented in Supplementary Fig. . e Western blotting showing the expression of Dnmt1 in whole cells (Total), mitochondria (Mito.), and cytoplasm (Cyto.) of MC-38 cells, with or without Acads knockdown. f qPCR showing the mRNA levels of Dnmt1 in MC-38 cells with or without Acads knockdown ( n = 3 per group). g Western blot analysis showing the abundance of Dnmt1 in mitochondria (mito.) and whole-cell lysates (WCL) of MC-38 cells following 24 h of treatment with MG132 (5 μM). h Western blot analysis demonstrating the level of Dnmt1 ubiquitination in immunoprecipitated MC-38 cells via an anti-Dnmt1 antibody. Immunofluorescence assay demonstrating the levels of 5-mC in MC-38 cells with or without Acads knockdown ( i , scale bar 20 μm), with the quantification of the mean fluorescence intensity of 5-mC shown in ( j ) ( n = 4 per group). k Whole-cell DNA methylation sequencing showing the levels of whole-genome methylation in MC-38 cells, with or without Acads knockdown. l ELISA results showing 5-mC methylation in mtDNA following its extraction from the purified mitochondria of Acads knockout and control MC-38 cells ( n = 3 per group). m qPCR results showing the relative levels of cytosolic mtDNA ( ND1 ) in MC-38 cells with or without mitochondrial Dnmt1 overexpression (Lv-mito- Dnmt1 , OE) ( n = 9 per group). n Western blotting showing the protein levels of the Cgas-Sting pathway in MC-38 cells with or without mitochondrial Dnmt1 overexpression. o qPCR results showing the relative mRNA levels of Ifn-β and Oasl1 in MC-38 cells with or without mitochondrial Dnmt1 overexpression ( n = 6 per group). p Histogram of tumor weight and representative image illustrating the progression of tumors in subcutaneous models established with MC-38 cells with or without mitochondrial Dnmt1 overexpression ( n = 4 per group). q qPCR results showing the relative levels of cytosolic mtDNA in MC-38 cells with or without Acads knocked down following 48 h of treatment with 5 μM decitabine ( n = 6 per group). Representative image ( r ) and histogram of tumor weight ( s ) illustrating the impact of decitabine on the progression of tumors in subcutaneous models established with Acads -knockdown MC-38 cells. During this period, decitabine (1.1 mg/kg) was administered via intraperitoneal injection every two days after the indicated cells were seeded ( n = 6 per group). ( t ) qPCR showing the relative levels of cytosolic mtDNA in MC-38 cells with or without CRISPR/Cas9-based mito- Dnmt1 knockout. No. 51 and 58 were screened from two mito- Dnmt1 knockout cell lines ( n = 4 per group). Representative image ( u ) and histogram of tumor weight ( v ) illustrating the progression of tumors in subcutaneous models established with MC-38 cells with or without specific knockout of mito-Dnmt1 ( n = 5 per group). The data are shown as the means ± SDs; n.s stands for not significant; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In brief, 4 μg of specific antibodies against Acads (#16623-1-AP, Proteintech) or Dnmt1 (#NB100-56519AF594, Novus Biologicals) and their respective IgGs were preincubated with magnetic beads (Sera-Mag SpeedBead Protein A/G, 17152104010150, GE) from a 40 μL suspension at 4 °C overnight.

Techniques: Methylation, Liquid Chromatography with Mass Spectroscopy, Lysis, Incubation, Magnetic Beads, Control, Western Blot, Blocking Assay, Amplification, Immunofluorescence, Staining, Imaging, Confocal Microscopy, Negative Control, Expressing, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Fluorescence, DNA Methylation Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Extraction, Purification, Knock-Out, Over Expression, Injection, CRISPR

Journal: Signal Transduction and Targeted Therapy

Article Title: Short-chain acyl-CoA dehydrogenase initiates mtDNA demethylation and leakage to fuel antitumor immunity in colorectal cancer

doi: 10.1038/s41392-026-02675-8

Figure Lengend Snippet: The ACADS-DNMT1-STING axis is correlated with the TME and CRC progression in human patients. a Representative H&E and immunohistochemistry images of ACADS in paracancerous and colon cancer tissues from 77 CRC patients (scale bar, 50 μm). b Histogram showing the H-scores of ACADS in 77 CRC and paracancerous tissues. c Kaplan‒Meier analysis of the survival curves of CRC patients with high and low ACADS expression from the microarray. d Histogram showing the H-scores of ACADS in different stages of CRC measured via a tissue microarray (paracancer from 77 CRC patients, stage 1 from 13 patients, stage 2 from 37 patients, and stage 3/4 from 27 patients). e Representative multiple immunohistochemistry images of DNMT1, ACADS, and HSP60 in CRC and paracancerous tissues from the microarray (scale bar, 50 μm). Histogram showing the percentage of mito-DNMT1-positive areas ( f ) and mito-DNMT1-positive cells ( g ) in CRC and paracancerous tissues from the microarray ( n = 77 patients). h Correlations between mito-DNMT1 and ACADS levels in CRC tissues measured via a tissue microarray. i Correlation of DNMT1 and ACADS protein levels in CRC tissues obtained from the CPTAC database ( n = 197). j The correlation of DNMT1 and ACADS mRNA levels in CRC tissues obtained from the TCGA-COAD database ( n = 434). k The correlation heatmap illustrates the relationships among ACADS, DNMT1, STING signaling, and various immune cells derived from the immune infiltration analysis results obtained via the xCell algorithm for these genes. Dot plots illustrating the correlation in expression between ACADS and either STING ( l ) ( n = 512) or IRF3 ( m ) ( n = 512), utilizing expression data from the TCGA-COAD cohort. n Low expression of ACADS was significantly correlated with poor immunotherapy benefit. The IPS score (ctla4: neg; pd1: pos) represents the IPS score when receiving only anti-PD1 therapy, the IPS score (ctla4: pos; pd1: neg) represents the IPS score when receiving only anti-CTLA-4 therapy, and the IPS score (ctla4: pos; pd1: pos) represents the IPS score when receiving both anti-CTLA-4 therapy and anti-PD1 therapy. The data are shown as the means ± SEMs, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In brief, 4 μg of specific antibodies against Acads (#16623-1-AP, Proteintech) or Dnmt1 (#NB100-56519AF594, Novus Biologicals) and their respective IgGs were preincubated with magnetic beads (Sera-Mag SpeedBead Protein A/G, 17152104010150, GE) from a 40 μL suspension at 4 °C overnight.

Techniques: Immunohistochemistry, Expressing, Microarray, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Short-chain acyl-CoA dehydrogenase initiates mtDNA demethylation and leakage to fuel antitumor immunity in colorectal cancer

doi: 10.1038/s41392-026-02675-8

Figure Lengend Snippet: Hypericin targets ACADS for immune activation and CRC therapy. a Schematic diagram of structure-based virtual screening for Acads-specific binders. b 2D (upper) and 3D (below) views of hypericin with Acads. The light blue cartoon represents Acads. c Three cell lines (MC-38, CT-26, and HCT-116) were treated with varying concentrations of hypericin (0, 5, and 10 µM) for 24 h. The protein level of Acads was then analyzed by Western blotting, with β-actin serving as the loading control. d SPR assay showing responses measured in the resonance unit (RU) of the ACADS protein (chip-coupled) to hypericin. e Western blotting showing the expression of Dnmt1 and Acads in whole cells (total) and mitochondria (Mito.) of MC-38 cells after 24 h of treatment with hypericin (10 µM). f , g qPCR showing the relative levels of cytosolic mtDNA ( ND1 and D-loop ) in MC-38 cells after 24 h of treatment with hypericin (10 µM, n = 4 per group). h Western blotting showing the protein levels of Cgas-Sting signaling pathway proteins (Cgas, Sting, Tbk1, and Irf3) in MC-38 cells following 24 h of treatment with hypericin (10 μM). i MC-38 cells with or without Acads knockdown were treated with hypericin (10 µM) or vehicle control for 24 h. Whole‑cell lysates were then collected and analyzed by qPCR to quantify the expression levels of the downstream Cgas-Sting effector Ifn-β , with β-actin serving as the loading control. j Experimental scheme of hypericin administration in MC-38 tumor‑bearing mice. k , l Representative image and histogram of tumor volume and weight ( l ) illustrating the impact of hypericin (10 mg/kg) on the progression of tumors in a subcutaneous mouse model established with MC-38 cells ( n = 5 per group). During this period, hypericin was first intraperitoneally injected (i.p.) on the 6th day after tumor inoculation. m Flow cytometry analysis showing the changes in MDSCs, macrophages, total T cells, CD8 + T cells, CD4 + T cells, IFNγ + T cells, and Tregs in subcutaneous tumors established by MC-38 cells, with or without 24 h of treatment with hypericin (10 mg/kg) ( n = 6 per group). n , o Flow cytometry analysis of the changes in total T cells (CD3 + ) and IFNγ + T cells (CD3 + IFNγ + ) in a mixture of single cells digested from CRC patients after 24 h of treatment with vehicle or hypericin (10 μM) ( n = 3 per group). p Schematic illustration of the mechanism by which ACADS deficiency promotes the progression of CRC. The data are shown as the means ± SDs; n.s stands for not significant; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In brief, 4 μg of specific antibodies against Acads (#16623-1-AP, Proteintech) or Dnmt1 (#NB100-56519AF594, Novus Biologicals) and their respective IgGs were preincubated with magnetic beads (Sera-Mag SpeedBead Protein A/G, 17152104010150, GE) from a 40 μL suspension at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Control, SPR Assay, Expressing, Knockdown, Injection, Flow Cytometry