Journal: International Journal of Molecular Medicine

Article Title: Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1-mediated mitophagy

doi: 10.3892/ijmm.2025.5693

Figure Lengend Snippet: HPV E7 induces mitophagy in keratinocytes (A) Mitophagy in NHEKs overexpressing HPV11/16 E7 was ascertained through transmission electron microscopy. The yellow arrows indicate mitochondrial autophagosomes. The number of mitochondrial autophagosomes was statistically analyzed and a total of 100 cells were analyzed per experiment. Intracellular mitophagy in NHEKs overexpressing (B) HPV11 E7 and (C) HPV16 E7 was analyzed with GFP-LC3 and Mito-Tracker Red CMXRos. The yellow dots indicate co-localized molecules. The ratio of yellow dots to all dots was statistically analyzed and a total of 100 cells were analyzed per experiment. Alterations in mitophagy-related proteins and pathways were detected by western blotting in NHEKs overexpressing (D) HPV11 E7 and (F) HPV16 E7. All proteins were normalized to the loading control GAPDH. Expression levels of mitophagy-related proteins and pathways were investigated in NHEKs overexpressing (E) HPV11 E7 or (G) HPV16 E7 and stimulated with the mitophagy inhibitor CsA. * P<0.05, ** P<0.01. BNIP3, Bcl-2 and adenovirus E1B 19 kDa-interacting protein 3; BNIP3L, BNIP3-like; CsA. CsA, cyclosporine A; NC, negative control; HPV, human papillomavirus; NHEK, normal human epidermal keratinocyte; PINK1, PTEN-induced kinase 1.

Article Snippet: The experimental results indicated that HPV11/16 E7 may activate the PINK1/Parkin and BNIP3/BNIP3L pathways, enhance LC3 expression, suppress p62 expression and consequently promote mitophagy in keratinocytes; however, this phenomenon was inhibited by the mitophagy inhibitor cyclosporine A (cat. no. HY-B0579; MedChemExpress; 5 μ mol/l, 12 h, 37°C) ( ).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Control, Expressing, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1-mediated mitophagy

doi: 10.3892/ijmm.2025.5693

Figure Lengend Snippet: HPV E7 activates mitophagy in keratinocytes through modulating the expression of HTRA1. (A) Transmission electron microscopy was adopted to observe the mitophagy status within HPV11/16 E7-overexpressing NHEKs with HTRA1 knockdown. The yellow arrows indicate mitochondrial autophagosomes. The number of mitochondrial autophagosomes was statistically analyzed and a total of 100 cells were analyzed per experiment. Western blotting was employed to detect the expression of mitophagy-related proteins and pathways in NHEKs with overexpression of (B) HPV11 E7 and (D) HPV16 E7 after HTRA1 knockdown. Co-localization of GFP-LC3 and Mito-Tracker Red CMXRos was employed to observe mitophagy within (C) HPV11 E7-overexpressing and (E) HPV16 E7-NHEKs with HTRA1 knockdown. The yellow dots represent the co-localized areas. The ratio of yellow dots to all dots was calculated and a total of 100 cells were analyzed for each experiment. All proteins were normalized to the loading control GAPDH. * P<0.05, ** P<0.01. BNIP3, Bcl-2 and adenovirus E1B 19 kDa-interacting protein 3; BNIP3L, BNIP3-like; CTRL, control; HPV, human papillomavirus; HTRA1, high-temperature requirement A serine peptidase 1; NHEK, normal human epidermal keratinocyte; PINK1, PTEN-induced kinase 1; sh, short hairpin.

Article Snippet: The experimental results indicated that HPV11/16 E7 may activate the PINK1/Parkin and BNIP3/BNIP3L pathways, enhance LC3 expression, suppress p62 expression and consequently promote mitophagy in keratinocytes; however, this phenomenon was inhibited by the mitophagy inhibitor cyclosporine A (cat. no. HY-B0579; MedChemExpress; 5 μ mol/l, 12 h, 37°C) ( ).

Techniques: Expressing, Transmission Assay, Electron Microscopy, Knockdown, Western Blot, Over Expression, Control

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Western blot validation of mCherry–EGFP–LC3 NMR stable over-expression in NMR skin fibroblast lines. Cell lysates from single-cell–derived colonies were probed with antibodies against LC3 ( A ) and EGFP ( B ). Both antibodies detected doublet bands at approximately 80–82 kDa, consistent with the predicted molecular mass of the mCherry–EGFP–LC3 NMR fusion protein in the non-lipidated (LC3-I; top band) and lipidated (LC3-II, bottom band) forms. Markers are shown in lane M and individual colonies are labelled C1-C6. C ) mTOR inhibition by PP242 enhances autophagic flux in NMR skin fibroblasts. Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expressing cells, either untreated or treated with PP242 (2 μM or 4 μM) for 24 h. PP242 treatment induces a shift from the appearance of autophagosomes (mCherry⁺/EGFP⁺ (yellow)) to autolysosomes (mCherry⁺/EGFP⁻ (red)). Scale bar is 10 μm. D) Bafilomycin A1 (Baf A1) impairs autophagic flux in NMR skin fibroblasts. Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expressing cells, either untreated or treated with Baf A1 (166 nM, 250 nM, or 333 nM) for 24 h. Baf A1 treatment results in accumulation of mCherry⁺/EGFP⁺ (yellow) puncta, consistent with impaired autophagic flux. In C) and D) the nucleus is stained with Hoescht (blue).

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Western Blot, Biomarker Discovery, Over Expression, Single Cell, Derivative Assay, Inhibition, Expressing, Staining

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: A ) Representative live-cell confocal images of HeLa cells and NMR skin fibroblasts stably expressing mCherry–EGFP–LC3 under basal-level culture conditions. The nucleus is stained with Hoescht (blue). Few puncta are present in the HeLa cells, and those that are correspond to mCherry + /EGFP - (red). In contrast, NMR skin fibroblasts display more puncta with a mixture of mCherry + /EGFP - (red) and mCherry + /EGFP + (yellow). B ) Quantification of LC3 puncta density in HeLa and NMR skin fibroblasts, normalised to cell area (puncta per μm²). Ten individual cells per cell line were analysed, sampled from four independent fields of view. Statistical significance was assessed using a two-tailed unpaired t-test. ****P < 0.0001. C ) Quantification of LC3 puncta diameter in HeLa cells and NMR skin fibroblasts. Twenty individual LC3 puncta per cell line were analysed, sampled from four independent fields of view. Statistical analysis was performed using a two-tailed unpaired t-test. n.s. - not significant.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Stable Transfection, Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: A) Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expression in cells either untreated or treated with increasing concentrations of CQ (40, 60 and 100 μM) for 4 h. The nucleus is stained with Hoescht (blue). CQ-treated cells exhibited predominantly mCherry⁺/EGFP⁺ (yellow) puncta, consistent with impaired lysosomal degradation. Brightfield panels show the appearance of the cytoplasmic vacuolation with increasing CQ concentrations. B ) WB analysis of mCherry–EGFP–LC3 NMR protein levels from cells either untreated or treated with increasing concentrations of CQ (10 μM, 20 μM, 40 μM, or 60 μM) for 24 h. CQ treatment resulted in an accumulation of LC3-II relative to LC3-I. ( C ) Quantification of LC3-II/LC3-I ratios derived from densitometric analysis. CQ treatment significantly increases LC3-II accumulation relative to controls. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Representative live-cell confocal images monitoring mCherry–EGFP–LC3 NMR following CQ treatment. The nucleus is stained with Hoescht (blue). Cells were treated with 40 μM CQ for 16 h or 24 h, as indicated. In the control (basal level), LC3-positive puncta are observed as mCherry⁺/EGFP⁻ (red) or mCherry⁺/EGFP⁺ (yellow) structures. After 16 h of CQ treatment, additional LC3-positive structures emerge, including mCherry⁺/EGFP⁺ ring-like structures associated with the surface of large cytoplasmic vacuoles (pink arrow) and mCherry⁺/EGFP⁺ ring-like structures enclosing red puncta (white arrow). Following 24 h of CQ treatment, the abundance of LC3-labelled vacuoles increases further.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expression in cells left untreated, treated with CQ (40 μM) for 24 h, or allowed to recover for 4 h or 24 h following CQ removal, as indicated. The nucleus is stained with Hoescht (blue). After 24 h of CQ treatment, LC3-positive structures predominantly appear as mCherry + /EGFP + (yellow) puncta and LC3-decorated ring-like vacuolar structures. Following the removal of CQ from the media, progressive reorganisation of LC3-labelled structures is observed. At 4 h of recovery, smaller mCherry + /EGFP - LC3 puncta and mCherry + /EGFP + structures are frequently observed, and ring-like structures are less apparent. By 24 h of recovery, LC3 labelling is no longer associated with vacuoles, and the majority of LC3 puncta exhibit a distribution comparable to untreated control cells.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Expressing, Staining, Control

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a KEGG pathway enrichment of DEGs in ZIKV-infected U251 cells transfected with HA-DISC1 (1 μg) plasmid compared with the control group transfected with pcDNA. b KEGG pathway enrichment of differentially expressed proteins identified by IP/MS analysis in U251 cells overexpressing HA-DISC1 (5 μg) plasmid compared with the control group transfected with pcDNA. c Chord diagram illustrates the proteins identified in the IP/MS analysis. d U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h followed by ZIKV infection. The protein levels of AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot at 0, 12, 24, and 36 h post-infection. e U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, rapamycin (25 μM), and MHY1485 (10 μM) for 24 h. ZIKV E expression was assessed by Western blot. f U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, autophagosome inhibitor 3-MA (1 mg/ml), and lysosome inhibitor CQ (100 μM) for 24 h. ZIKV E expression was assessed by Western blot. KEGG pathway enrichment analyses in ( a , b ) were performed using a one-tailed Fisher’s exact test. Images in ( d − f ) are one representative experiment out of three independent replicates with similar results ( n = 3). Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Infection, Transfection, Plasmid Preparation, Control, Protein-Protein interactions, Western Blot, Expressing, One-tailed Test

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Conserved LIR motifs (amino acids 210 FSFI 213) in DISC1 were highlighted in yellow, which were mutated into AAAA in DISC1 mutant. b 293T cells were co-transfected with HA-DISC1 (5 μg) or HA-DISC1 mutant (5 μg) and GFP-LC3 (5 μg) plasmids for 48 h. Cellular lysates were subjected to immunoprecipitation with anti-Flag or anti-HA magnetic beads and Western blot assays using the indicated antibodies. c − e U251 cells were transfected with HA-DISC1 (1 μg) or HA-DISC1 mutant (1 μg) plasmids for 24 h, followed by ZIKV infection for 24 and 48 h. Viral mRNA levels, protein expression and titers were measured on D1 and D2 post-infection by qRT-PCR ( c ), Western blot ( d ), and plaque assays ( e ). Images in ( b , d ) are from one representative experiment out of three independent replicates with similar results ( n = 3). Data shown in ( c , e ) are from one representative experiment out of three independent replicates with similar results, each including 3 biological replicates per group ( n = 3). Data shown in ( c , e ) were confirmed to follow a normal distribution using the Shapiro-Wilk test, and statistical analysis was performed using two-tailed one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Infection, Expressing, Quantitative RT-PCR, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Primary cortical neurons were isolated from neonatal mice (P0–P1) and induced to differentiate in vitro. On day 7 of differentiation, neurons exhibiting neurite outgrowth were observed under bright-field microscopy. Confocal imaging showed neuronal nuclei stained with DAPI (blue), and endogenous Neun labeled with an anti-Neun antibody (red). Scale bar = 50 μm. b , c WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. Viral mRNA levels and titers were measured on D1 and D2 post-infection using qRT-PCR ( b ) and plaque assay ( c ). d WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. The protein levels of ZIKV E protein, DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot on D1 and D2 post-infection. e, f The ratios of pAMPKα to AMPKα ( e ) and pmTOR to mTOR ( f ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8d. g − i Quantification of P62 ( g ), LC3A/BⅠ ( h ) and LC3A/BⅡ ( i ) protein levels relative to GAPDH at each time point in Fig. 8d. j , k Placentas ( j ) and fetal heads ( k ) from non-infected and ZIKV-infected WT and Disc1 KD mice were collected on E13.5. The protein levels of DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot. l − o The ratios of pAMPKα to AMPKα ( l, m ) and pmTOR to mTOR ( n , o ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8j ( l, n ) and Fig. 8k ( m , o ). p − s Quantification of P62 ( p , q ) and LC3A/BⅡ ( r , s ) protein levels relative to GAPDH at each time point in Fig. 8j ( p , r ) and Fig. 8k ( q , s ). Data shown in ( b , c ) are from one representative experiment out of three independent replicates with similar results, each including 3 biological replicates per group ( n = 3). Images in ( a , d , j – k ) are from one representative experiment out of three independent replicates with similar results ( n = 3). Data shown in ( e − i , l − s) are collected from three independent experiments ( n = 3). Data shown in ( b − c , e − i , l − s ) were confirmed to follow a normal distribution using the Shapiro-Wilk test, and statistical analysis was performed using Student’s unpaired two-tailed t test ( b , c ) or two-tailed one-way ANOVA followed by Tukey’s multiple comparison test ( e − i , l − s ). Data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Isolation, In Vitro, Microscopy, Imaging, Staining, Labeling, Incubation, Infection, Quantitative RT-PCR, Plaque Assay, Western Blot, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a 6-8-weeks-old WT or Disc1 KD mice were treated with 2 mg MAR1-5A3 on the day prior to infection and then intraperitoneally (i.p.) inoculated with PBS or 1 × 10 6 PFU of ZIKV. Mouse brains were collected on D6 post-infection. Representative H&E-stained sections of the hippocampal DG and CA3 regions are shown. Black arrows indicate shrunken, hyperchromatic neurons with indistinct boundaries Scale bar = 100 μm. b Representative immunofluorescence-stained sections of the hippocampal DG region. Cell nuclei were stained using DAPI (blue). Endogenous Neun was labeled with anti-Neun antibody (red). Endogenous LC3A/B was labeled with anti-LC3A/B antibody (yellow). Scale bar = 100 μm. Representative images in ( a , b ) are from one independent experiment, including 6 mice per group ( n = 6), consisting of 3 males and 3 females.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Infection, Staining, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Neonatal mice were intracranially injected with 200 PFU of ZIKV GZ01 or an equal volume of PBS into the λ point of the brain on postnatal day 2 (P2). Mouse brains were collected on D6 post-infection. Representative H&E-stained sections of the hippocampal CA1 region are shown. Black arrows indicate shrunken, hyperchromatic neurons with indistinct boundaries. Orange arrows indicate nuclear pyknosis and fragmentation. Scale bar = 100 μm. b Representative immunofluorescence-stained sections of the hippocampal DG region. Cell nuclei were stained using DAPI (blue). Endogenous Neun was labeled with anti-Neun antibody (red). ZIKV E protein was labeled with anti-E antibody (green). Endogenous LC3A/B was labeled with anti-LC3A/B antibody (yellow). Scale bar = 100 μm. Representative images in ( a , b ) are from one representative experiment out of two independent experiments with similar results, each including 4 mice per group ( n = 4), consisting of 2 males and 2 females.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Injection, Infection, Staining, Immunofluorescence, Labeling

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: Effects of the DNA-damaging cytolethal distending toxin ( Cj -CDT) on autophagy HCT116 cells were incubated in McCoy’s 5A + 10% FBS at 37°C and under 5% CO 2 in the absence or presence of Cj -CDT (10 nM). After 24 h, cell monolayers were further incubated with nutrient-rich McCoy’s 5A + 10% FBS ( A – G ), autophagy inducing nutrient-poor starvation medium (EBSS, Earl’s Balanced Salt Solution) ( A – E ), CCCP (25 μM) in McCoy’s 5A + 10% FBS ( F ), or rapamycin (20 μM) in McCoy’s 5A + 10% FBS ( G ). After 4 h, cells were imaged using fluorescence microscopy for total LC3-associated fluorescence signal intensity and total LC3 puncta ( A and D ), or, analyzed by immunoblot analysis for relative levels of LC3-II, γ-H2AX, and β-actin ( B , C , and E–G ). White scale bars from representative images indicate 10 μm ( A and D ). Images are representative of those collected from three biologically independent experiments ( n = 3) collected at 40× magnification. ( A and D ) Densitometric analyses of immunoblots ( B, C, and E – G ) collected from 3 biologically independent experiments ( n = 3) were combined. Error bars represent standard deviations. Statistical analyses of the data were conducted using one-way Anova, followed by Dunnett’s post hoc test ( A – C ), or a two-tailed, unpaired Student’s t test ( D – G ). Data are represented as mean ± SD. p < 0.05 indicating statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Incubation, Fluorescence, Microscopy, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: Cj -CDT-dependent effects on autophagolysosomal turnover HCT116 cells within nutrient-rich McCoy’s 5A + 10% FBS medium, were incubated at 37°C in the absence (PBS pH 7.4) or presence of Cj -CDT (10 nM). After 24 h, the cell monolayers were washed 3 times with PBS. The monolayers were then further incubated at 37°C and under 5% CO 2 , in the absence of toxin, with either EBSS or McCoy’s 5A + 10% FBS, as well as in the absence or presence of pepstatin A (10 μg/mL) and E−64d (10 μg/mL) ( A – C ), or, in the absence or presence of chloroquine (60 μM) and in the presence of actinomycin D (4 μM) ( D ). Relative levels of cellular LC3 were examined by fluorescence microscopy ( A ). White scale bars in representative images indicate 10 μm ( A ). Relative levels of LC3-II ( B ), mKeima ( C ), and SQSTM/p62 ( D ) were examined by immunoblot analysis. Images collected at 40× magnification ( A ) and immunoblots ( B to D ) are representative of those collected from three biologically independent experiments ( n = 3). Data from 3 biologically independent experiments ( n = 3) were combined and are shown ( A – D ). Error bars represent standard deviations of data combined from 3 biologically independent experiments ( n = 3). Statistical analyses of the data were conducted using unpaired, two-tailed, Student’s t test ( A and B ), or, two-way ANOVA, followed by Fisher’s LSD post hoc test ( C and D ). Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Incubation, Fluorescence, Microscopy, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: Depletion of LC3-I is causal for the reduction in cellular LC3-II in cells exposed to Cj -CDT HCT116 cells transfected with RedTrackCMV or RedTrackCMV-LC3B were incubated with Cj -CDT (10 nM) or Bleocin (10 μg/mL) ( A – C ). After 24 h, cell lysates were further incubated for 3 h in the absence ( A and C ) or presence ( B and C ) of the autophagy inducer, CCCP (25 μM), and then analyzed by immunoblot analysis for LC3-I and LC3-II ( A and B ), or, γ-H2AX ( C ). Densitometric analyses of immunoblots collected from 3 biologically independent experiments ( n = 3) were combined. Error bars represent standard deviations. Statistical analyses of the data were conducted using one-way Anova, followed by Dunnett’s post hoc test ( A and B ). Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Transfection, Incubation, Western Blot

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: The DNA damage response is important for CDT-dependent reduction in both cellular LC3-I and LC3-II levels HCT116 ( p53 +/+ ) cells ( A – E ) or HCT116 ( p53 −/− ) cells ( C ) were preincubated with KU-55933 (10 μM) ( A and B ), and then in the absence or presence of Cj -CDT (10 nM) ( A – C ), inactive CDT (containing Cj -CdtB H157G, 10 nM) ( A – C ), or Bleocin (10 μg/mL) ( D ). Alternatively, cell monolayers were incubated in the absence or presence of Nutlin-3 (20 μM) ( D and E ). After 24 h, monolayers were further incubated with CCCP (25 μM) ( A, C, and E ). Lysates were analyzed by immunoblot analysis for relative levels of LC3-I ( A, C, and E ), LC3-II ( A, C, and E ), β-actin ( A and C ), total p53 ( D ) p-p53 (S15) ( B – D ), and GAPDH ( B, D, and E ). The immunoblots are representative of those from 3 independent experiments ( n = 3). Densitometric analyses were combined from 3 independent experiments ( n = 3), and the relative levels of LC3-I, LC3-II, and p-p53 were calculated by dividing the intensity of immuno-specific bands corresponding to cellular LC3-I ( A, C, and E ), LC3-II ( A, C, and E ), and p-p53 ( B ) by the intensity of immuno-specific bands corresponding to controls β-actin ( A and C ) or GAPDH ( B and E ). The data represented by the black bars were each separately rendered relative to the data represented by the white bars, which were assigned an arbitrary value of 1.0. Error bars represent standard deviations. two-way ANOVA followed by Fisher’s LSD post hoc test ( A and C ), or two-tailed, unpaired Student’s t test ( B and E ) were used. Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Incubation, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: DNA damage-dependent suppression of autophagy is linked to the ubiquitination and depletion of LC3 by a proteasome-dependent mechanism MEF Atg5 +/+ and MEF Atg5 −/− cells were incubated in the absence or presence of Cj -CDT (10 nM) ( A ). After 48 h, MEF cells were further incubated with rapamycin (20 μM) ( A ). After 3 h in the presence of rapamycin, cell lysates were examined for LC3-I, LC3-II, and β-actin ( A ). HCT116 cells ( B – E ), either transfected ( C ), or, not transfected ( B – E ) with the pCMV-Myc-LC3 overexpression plasmid, were incubated with Cj -CDT (10 nM) ( B – D ), Bleocin (10 μg/mL) ( B ), or Nutlin 3 (20 μM) ( E ), and, in the presence or absence of MG132 (380 nM) ( B – D ) or TAK-243 (100 nM) ( C ). After 24 h, HCT116 monolayers were further incubated with CCCP (25 μM) ( B ), while Nutlin-treated monolayers were further incubated with MG132 (380 nM) ( E ). After 3 h in the presence ( B ) or absence ( B to D ) of CCCP, or 4 h in the presence of MG132 ( E ), cell lysates were examined for LC3-I ( A – E ), LC3-II ( A – E ), Myc-LC3B ( C ), ubiquitin ( C ), total p53 ( E ), and β-actin ( B – E ). Alternatively, Myc-LC3B was immunoprecipitated with Myc-specific antibodies, and ubiquitinated Myc-LC3B was detected using Myc-specific and ubiquitin-specific antibodies ( C ). Intrinsic cellular LC3B was immunoprecipitated using LC3-specific antibodies,and ubiquitinated LC3B was detected using ubiquitin-specific antibodies ( D and E ). Immunoblots are representative of data collected from at least 3 biologically independent experiments ( n = 3). Densitometric analyses were combined from three independent experiments. The data shown by the black (filled) bars were each separately rendered relative to the data represented by the white (empty) bar, which was assigned an arbitrary value of 1.0. Error bars represent standard deviations. Statistical analyses of the data were conducted using two-tailed, unpaired Student’s t test ( A and E ) or two-way ANOVA, followed by Fisher’s LSD post hoc test ( B – D ). Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Ubiquitin Proteomics, Incubation, Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: DNA damage-dependent reduction in both cellular LC3-I and LC3-II levels is reversed by impairing the ATM-mediated stabilization of p53 HCT116 cells were incubated for 24 h with Cj -CDT (10 nM), or Bleocin (10 μg/mL). After 24 h, monolayers were further incubated in the absence or presence of the autophagy inducing compound CCCP (25 μM) ( B ), as well as the absence or presence of the ATM inhibitor, KU-55933 (10 μM). After an additional 3 h, cell lysates were examined, using immunoblot analyses, for total p53 ( A ), p-p53 (Ser15) ( A ), LC3-I ( B ), LC3-II ( B ), and β-actin ( A and B ). The immunoblots shown are representative from 3 biologically independent experiments ( n = 3). Densitometric analyses were combined from 3 immunoblots ( B ), and the relative levels of cellular LC3-I or LC3-II levels were calculated. The data shown by the black (filled) bars were each separately rendered relative to the data represented by the white (empty) bar, which was assigned an arbitrary value of 1.0. Error bars represent standard deviations of data combined from 3 biologically independent experiments ( n = 3). Statistical analyses of the data were conducted using two-way ANOVA, followed by Fisher’s LSD post hoc test. Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Incubation, Western Blot

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: Model of the proposed p53-proteasome-LC3 axis for regulating the autophagic response in DNA damaged cells Autophagy suppression results from the proteasomal degradation of LC3 within DNA damaged cells as a mechanism for maintaining viability during the restoration of genome integrity ( A – D ). Upon DNA damage, DDR activation leads to the stabilization of p53, which is required for the ubiquitination of LC3 ( A ), thereby targeting LC3 for proteasome degradation ( B ). Available LC3 pools for autophagosome maturation are diminished leading to the production of fewer mature autophagosomes ( C ). Fewer mature autophagosomes results in reduced autophagic flux and preservation of pro-survival factors ( D ).

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Activation Assay, Ubiquitin Proteomics, Preserving

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: After 12 h, cells were transfected with a Myc-tagged LC3 ectopic expression plasmid (pCMV-myc-LC3; #24619, Addgene) using Lipofectamine 3000 transfection reagent (#L300015, Thermo Fisher Scientific, USA).

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis