Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A A3C pulldown and immunoblot analysis of H1299 cell lysates with or without gemcitabine treatment. * Shorter exposure time. B Venn diagram illustrated the intersection of A3C interactome with or without gemcitabine treatment. C Enrichment analysis of pathways based on Gene Ontology (GO). D A3C interactome was screened by mass spectrometry-based proteomics. The table showed the representative highest scored peptides that physically bind to A3C. Our AP-MS dataset was verified by published proteomic data (Cheung group, 2018) from RNA/DNA hybrids immunoprecipitation (DRIP) located at the BAMBI promoter and DPP9 3’ UTR. E A Venn diagram illustrated the intersection of A3C interactome (291, after removed keratins and other contaminants) with 803 proteins that interact with RNA/DNA hybrids published by Cheung group. Molecular Complex Detection (MCODE) was executed to identify key modules in the protein-protein interaction (PPI) network of A3C-hybrids binding. DDX5, DDX3X and DHX9 were highlighted in the highly interconnected clusters. F R-loop-dependent A3C-DDX5 interaction. H1299 cells grown on 10 cm dishes were co-transfected with 5 μg FLAG-tagged A3C and 5 μg HA-tagged DDX5, along with either 5 μg wild-type RNase H1 (ppyCAG_RNaseH1), the catalytically inactive D210N mutant, or an empty vector (pcDNA3.1). Cell lysates were analyzed by immunoprecipitation using anti-FLAG antibodies followed by immunoblots with the indicated antibodies. Input indicates 20% of pre-immunoprecipitated samples. G The full-length (FL), N-terminal (ΔΝ), C-terminal (ΔC) or both (ΔΝC) truncated forms of DDX5. H HEK-H293T cells were transfected with full-length (FL) HA-DDX5, or the DDX5 truncation mutants for 24 h. Lysates were incubated with immobilized HA. Affinity precipitated exogenous DDX5 was detected with anti-HA antibody, with the relative levels in the lysates also shown. I Display of the Predicted Aligned Error (PAE) for the AlphaFold-predicted A3C/DDX5 complex. DDX5 and A3C chains are labeled respectively in cyan and orange. A dark green tile designates a good prediction, and the region of DDX5 that is expected to be in proximity to A3C includes residues 50-100, while for A3C, the residue range is 30-180. Structure-based protein interaction interface analysis between A3C and DDX5. Image represents the predicted A3C-DDX5 complex, where interaction interface and hotspot residues are labeled. J Structure-based protein interaction interface between A3C (PDB ID: 3vow) and DDX5 (PDB ID: 4a4d) where interaction region and two hotspot residues (Tyr59/Tyr97) are labeled.

Article Snippet: For tumor xenografts, frozen tissue sections were fixed with cold acetone for 10 min and were blocked with 5% BSA containing 0.3% TritonX-100 for 2 h. Sections were incubated with a 1:100 dilution of S9.6 antibody (Kerafast, cat. no. ENH001) and DDX5 (CST, cat. no. 9877), followed by incubation with a 1∶200 dilution of Alexa Fluor 647 or 488-conjugated goat anti-mouse/rabbit secondary antibody and ProLong Gold Antifade Mountant with DAPI, and then observed under a confocal microscope with 40× magnification objective lens (Zeiss LSM 880 NLO).

Techniques: Western Blot, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Binding Assay, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Labeling, Residue

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Immunofluorescent staining with the S9.6 antibody showed the R-loop formation (green foci) in response to gemcitabine or/and Chk1 inhibitors in H1299 cells expressing A3C, DDX5 or their control counterparts (pCMV6). Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm. Quantification of the number of S9.6 foci per nucleus and the percentage of nucleus displaying more than 3 foci is plotted. Each bar represents the mean percentage (%) ± SD by three biologically triplicate experiments and at least 40 cells per group were analyzed for each experiment. Comparisons test was performed by one-way ANOVA followed by post-hoc Tukey’s test. * P = 0.0112, *** P < 0.001. B H1299 cells were treated with gemcitabine or/and Chk1 inhibitors for 6 and genomic DNA in a dilution of 400 ng/μL was extracted for assessing R-loop levels. Nuclear samples digested with or without RNase H (0.5 U) were loaded onto nylon membranes (2.5 μL per dot) and probed with S9.6 antibody. C Dot-blot analysis for A3C or DDX5-overexpressing cells treated with gemcitabine or/and Chk1 inhibitors. D H1299 cells were transfected with DDX5 (DDX5 OE ) or empty vector (DDX5 NC ) for 48 h, and further challenged with gemcitabine (1 μM) in the presence or absence of Chk1 inhibitors (AZD, 1 μM; MK, 10 μM) for another 6 h. Whole cell lysates were harvested for detecting the levels of phosphorylated RPA32 (Ser8), phosphorylated Chk1 (Ser345), phosphorylated histone H2AX on serine 139 (γH2AX), total RPA32 and Chk1. GAPDH was used as loading control. E A3C-overexpressing H1299 cells were transfected with DDX5 (DDX5 OE ) or empty vector (DDX5 NC ) for 48 h and challenged with gemcitabine (1 μM) for 6 h. Whole cell lysates were harvested for detecting the levels of phosphorylated Chk1 (Ser345) and Chk1. F EdU incorporation assay was performed in A3C OE /DDX5 NC and A3C OE /DDX5 OE H1299 cells. All the cells were stimulated with or without gemcitabine for 24 h. Data are expressed as the mean ± SD by triplicate assays. One-way ANOVA followed by post-hoc Tukey’s analysis was performed. ** P = 0.0098.

Article Snippet: For tumor xenografts, frozen tissue sections were fixed with cold acetone for 10 min and were blocked with 5% BSA containing 0.3% TritonX-100 for 2 h. Sections were incubated with a 1:100 dilution of S9.6 antibody (Kerafast, cat. no. ENH001) and DDX5 (CST, cat. no. 9877), followed by incubation with a 1∶200 dilution of Alexa Fluor 647 or 488-conjugated goat anti-mouse/rabbit secondary antibody and ProLong Gold Antifade Mountant with DAPI, and then observed under a confocal microscope with 40× magnification objective lens (Zeiss LSM 880 NLO).

Techniques: Staining, Expressing, Control, Dot Blot, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Further modulation of DDX5 expression in A3C-overexpressing H1299 cells for evaluating the functional interplay between A3C and DDX5 (overexpression, OE; knockdown, KD; NC, negative control). B A3C OE /DDX5 NC and A3C OE /DDX5 KD cells were co-treated with gemcitabine or/and Chk1 inhibitors for 6 h, and dot-blot analysis was performed for detecting R-loop levels using S9.6 antibody. Methylene blue was a loading control. C Drug sensitivity interaction between gemcitabine and two Chk1 inhibitors were evaluated in A3C OE /DDX5 NC and A3C OE /DDX5 KD H1299 cells using Combenefit and CompuSyn programs. * P = 0.0359, ** P = 0.0085, *** P < 0.001. D Comet assay in A3C OE /DDX5 NC and A3C OE /DDX5 KD H1299 cells. Data are expressed as the mean ± SD. One-way ANOVA followed by post-hoc Tukey’s analysis was performed (* P = 0.0279, *** P < 0.001). Scatter plots show all points, medians, and lower and upper quantiles. E For xenograft mouse model, A3C OE /DDX5 NC and A3C OE /DDX5 KD cells were inoculated subcutaneously into the right flank of nude mice. After developing tumors, the mice were divided into four cohorts (n = 6) and treated with either (1) vehicle (normal saline); (2) gemcitabine (100 mg/kg, i.p., Q3Dx5 or every third day for five doses). F Tumor size was recorded using caliper measurements. Tumor growth over time periods among four groups were compared by two-way repeated-measures ANOVA with the Greenhouse-Geisser correction and post-hoc analyses were performed by Šidák- adjusted multiple comparison tests. ** P = 0.0016 between gemcitabine-treated A3C OE /DDX5 KD xenografts and control xenografts. G Daily mean body weights were determined for all groups. H Representative fluorescence images of DDX5 (red) and S9.6 (green foci) staining positivity from xenograft tissues at 400× magnification. Nuclei were stained with DAPI. Scale bar represents 50 μm.

Article Snippet: For tumor xenografts, frozen tissue sections were fixed with cold acetone for 10 min and were blocked with 5% BSA containing 0.3% TritonX-100 for 2 h. Sections were incubated with a 1:100 dilution of S9.6 antibody (Kerafast, cat. no. ENH001) and DDX5 (CST, cat. no. 9877), followed by incubation with a 1∶200 dilution of Alexa Fluor 647 or 488-conjugated goat anti-mouse/rabbit secondary antibody and ProLong Gold Antifade Mountant with DAPI, and then observed under a confocal microscope with 40× magnification objective lens (Zeiss LSM 880 NLO).

Techniques: Expressing, Functional Assay, Over Expression, Knockdown, Negative Control, Dot Blot, Control, Single Cell Gel Electrophoresis, Saline, Comparison, Fluorescence, Staining