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

Journal: BMC genomics

Article Title: Autophagy is involved in the toxicity of the biocontrol agent GC16 against Tetranychus pueraricola (Acari: Tetranychidae) based on transcriptomic and proteomic analyses.

doi: 10.1186/s12864-025-11312-7

Figure Lengend Snippet: Fig. 6 GC16 induced autophagy in Sf9 cells. (A) Representative fluorescence staining images of the LC3 protein in Sf9 cells treated with GC16 or water (for control, designated as CK) for 24 h (scale bar = 20 μm). The region of the LC3 protein that displays positive staining is observed to be red in color. The staining of the nucleus is visible in blue with DAPI. Autophagic vacuoles were observed to be red in color. (B) Average fluorescence intensity of the LC3 protein in Fig. 6A. Data are represented as mean ± SE of three replicates (*P < 0.05, **P < 0.01, and *** P < 0.001 with compared to control). (C) Western blotting analysis for expression of autophagy marker LC3 in Sf9 cells treated with GC16 for 24 h. β-Actin was used as a loading control. The blots were cropped and in supplementary file the full-length blots/gels are presented. (D) A quantitative analysis of western blot was shown by LC3-II/LC3-I ratios in the down panel. All data are represented as mean ± SE of three experiments in triplicate (*P < 0.05, **P < 0.01, and *** P < 0.001 with compared to control)

Article Snippet: Sf9 cells were cultured on coverslips and treated with water (control) or 2.1 mg/mL GC16 for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 at 25 °C for 5 min, and then blocked (AR1009; BOSTER Biological Technology Co., Ltd., Wuhan, China) at 25 °C for 30 min. LC3 expression was detected by incubating the samples with primary antibodies against LC3 (AP0762, Bioworld Technology, Inc., USA) at 4 °C overnight and then with corresponding secondary antibodies (BA1032, BOSTER Biological Technology Co. Ltd, Wuhan, China) at 37 °C for 1 h. Subsequently, the samples were stained with DAPI (C1002, Beyotime Biotechnology, Shanghai, China) for 5 min, and covered with a sealing agent to prevent fluorescence quenching (0100- 01, Southern Biotechnology Associates, Inc., USA).

Techniques: Fluorescence, Staining, Control, Western Blot, Expressing, Marker