Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: P gingivalis inhibits mitophagy through disruption of ceramide, LC3, and Annexin A2 (ANXA2) binding (A) Confocal images of UMSCC1A cells infected/uninfected with P. gingivalis (100 MOI, 6 h) and treated with vehicle or LCL768 (30 μM, 2 h) were stained for LC3 (green) and Tom20 (red). (B) Confocal images of UMSCC1A cells infected/uninfected with P. gingivalis and treated with vehicle or LCL768, labeled for LAMP1 (green) and Tom20 (red). (C and D) Similarly, confocal images of ceramide (green) and LC3 (red), and LC3 (green) and P. gingivalis (red) are shown. Yellow shows colocalization. Images represent three independent experiments. Scale bars are 100 μm (throughout the manuscript unless specifically noted). Quantification of colocalization was estimated via Pearson’s correlation coefficient, completed with ImageJ Fiji software, and normalized to uninfected, untreated control. Values indicate mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E) Western blotting was used to detect the protein abundance of stably knocked down LC3 and transiently transfected tagged mutant or wildtype LC3 in UMSCC1A cells. Actin was used as the loading control. (F) UMSCC1A cells stably expressing shRNA against LC3 and transiently transfected with FLAG-wildtype LC3, V5-F52A LC3, or V5-G120A LC3 pcDNA3.1+ plasmids were labeled with ceramide antibody and tag antibody (FLAG or V5) for confocal microscopy. Quantification of colocalization was estimated using Pearson’s correlation coefficient and normalized to uninfected, untreated control using ImageJ Fiji software. Images represent three independent experiments. Data are means ± SD ( n = 3, ns, not significant, ∗∗∗∗ p < 0.0001). (G) UMSCC1A cells stably expressing shRNA against LC3 were transiently transfected with FLAG-wildtype LC3, V5-F52A LC3, or V5-G120A LC3 plasmids before infection with P. gingivalis (6 h, 100 MOI) and treatment with 30 μM LCL768 or vehicle for 3 h. Cells were dual labeled with P. gingivalis antibody and the appropriate tag antibody (FLAG or V5) for confocal microscopy. Quantification of colocalization, shown via Pearson’s correlation coefficient, are means ± SD ( n = 3, ns, not significant, ∗∗∗∗ p < 0.0001). (H) Live UMSCC1A cells were stained using Mtphagy dye (red) in which fluorescence intensity increases with mitophagy induction, following treatment with 5 μM cisplatin, 8 μM rapamycin, or 8 μM sodium selenite for 3 h. Images represent three independent experiments. The bottom panels show the quantification of Mtphagy fluorescence intensity using ImageJ Fiji software. Data are means ± SD ( n = 3, ∗∗∗∗ p < 0.0001). (I) UMSCC1A cells were transiently transfected with pHAGE-MtKeima plasmid, in which auto-fluorescent Keima protein conjugated to mitochondrial-targeting sequence (COX8) fluoresces green at neutral pH (>pH 6) and red at acidic pH (

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Disruption, Binding Assay, Infection, Staining, Labeling, Software, Control, Western Blot, Quantitative Proteomics, Stable Transfection, Transfection, Mutagenesis, Expressing, shRNA, Confocal Microscopy, Fluorescence, Plasmid Preparation, Sequencing

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: ANXA2 association with mitophagy markers (A) UMSCC1A cells were lysed for immunoprecipitation with LC3 (right) or species-matched IgG control antibody (left). Co-immunoprecipitation of ANXA2 is shown via western blot, and input controls were normalized to GAPDH. Images represent at least three independent experiments. Quantification of co-immunoprecipitated ANXA2 western blot bands from was normalized to uninfected, untreated control. Data are means ± SD ( n = 3, ∗ p < 0.05, ∗∗∗ p < 0.001). (B) Quantification of confocal images of UMSCC1A cells dual-labeled with LC3 and ANXA2 antibody, infected with P. gingivalis , and treated with LCL768. Pearson’s correlation coefficient was normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Confocal images of ceramide (green) and ANXA2 (red) in UMSCC1A cells infected with P. gingivalis and treated with LCL768 are shown. (D) Confocal images of Atg7 (green) and ANXA2 (red) in UMSCC1A cells infected with P. gingivalis and treated with LCL768. (E and F) Uninfected UM-SCC-1A cells were transfected with either sham or P. gingivalis FimA virulence factor proteins and treated with LCL768 or vehicle control. Confocal images of ceramide (green) and ANXA2 (red), and LC3 (green) and Tom20 (red), in uninfected cells are shown. Yellow shows colocalization. Images represent three independent experiments. Quantification of colocalization was estimated via Pearson’s correlation coefficient, completed with ImageJ Fiji software, and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Far western blotting analysis was conducted by running human recombinant ANXA2 (rANXA2) on a gel and testing direct protein-protein binding with recombinant P. gingivalis virulence factors (rFimA, rNDK). The top left blot shows direct binding between ANXA2 and FimA recombinant proteins, identified via FimA antibody. No direct binding between human recombinant ANXA2 and P. gingivalis recombinant NDK was observed in the bottom left blot. The top right blot confirms recombinant ANXA2 presence in the blot. The bottom right blot indicates that there is no cross-reactivity between recombinant ANXA2 and FimA antibody. (H) Immunofluorescence confirmation of wildtype and FimA-deficient (ΔFimA) P. gingivalis internalization into UM-SCC-1A cells with anti- P. gingivalis antibody is shown. Quantification of mean fluorescent intensity was completed with ImageJ Fiji software and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. Ns, not significant, ∗∗∗∗ p < 0.0001.

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Immunoprecipitation, Control, Western Blot, Labeling, Infection, Transfection, Software, Far Western Blot, Recombinant, Protein Binding, Binding Assay, Immunofluorescence

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: ANXA2 knockdown inhibits mitophagy (A and B) Immunoprecipitation with Tom20 or species-matched IgG antibody in uninfected UMSCC1A cells stably expressing shRNA against ANXA2 or scrambled control. The left panel shows input (pre-immunoprecipitation) ANXA2 and LC3 via western blotting, normalized to Actin control bands. The right panel shows co-immunoprecipitated LC3 in cells treated with LCL768 or vehicle control. Images represent at least three independent experiments. Quantification of co-immunoprecipitated LC3 western blot bands was normalized to scrambled shRNA expressing, untreated control. Data are means ± SD ( n = 3, ∗ p < 0.05, ∗∗∗∗ p < 0.0001). (C and D) Quantification of confocal images of uninfected UMSCC1A cells stably expressing shRNA against ANXA2 or scrambled control and treated with LCL768 or vehicle control. Colocalization between LC3 and Tom20 (C), or ceramide and Tom20 (D), was estimated via Pearson’s correlation coefficient and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E and F) Confocal images of uninfected UMSCC1A cells stably expressing shRNA against ANXA2 or scrambled control, treated with LCL768 or vehicle control and dual labeled with ceramide (green) and LC3 (red), or LAMP1 (green) and Tom20 (red) antibodies. (G) Quantification of confocal images in (E) and (F), shown via Pearson’s correlation coefficient. Values indicate mean ± SD of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01. (H and I) Proximity ligation assay was performed on uninfected UMSCC1A cells stably expressing shRNA against ANXA2 or scrambled control for ceramide and LC3, or ceramide and Tom20. Confocal images show DAPI nuclei staining (blue) and PLA signal (red) indicating interaction of targets within close proximity (<40 nm). (J) Quantification of the average number of PLA signal foci per cell in (H) and (I) was completed with ImageJ Fiji. Values indicate mean ± SD of n = 3 independent experiments. Ns, not significant, ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (K) Live, uninfected UMSCC1A cells stably expressing shRNA against ANXA2, LC3, or scrambled control were stained with Mtphagy (red) and LysoGreen dyes following treatment with LCL768 or vehicle control. Images represent three independent experiments. Yellow indicates colocalization. (L) Quantification of Mtphagy fluorescence intensity in (K) was done using ImageJ Fiji software. Data are means ± SD ( n = 3, ∗∗∗∗ p < 0.0001). (M) Live, uninfected UMSCC1A cells stably expressing shRNA against ANXA2, or scrambled control, were treated with increasing concentrations of LCL768 for 2 h and stained with trypan blue cell exclusion dye. Cell viability (percent live, unstained cells) is shown as means ± SD ( n = 3, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001).

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Knockdown, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Control, Western Blot, Labeling, Proximity Ligation Assay, Staining, Fluorescence, Software

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: Mutation of the projected ceramide-binding site on ANXA2 (E142A) inhibited ceramide-mediated mitophagy induction and FimA association (A) Trimolecular modeling of ANAX2, ceramide, and LC3 in MOE. (B) Docking of ceramide onto ANXA2. Bimolecular modeling of ANXA2 and ceramide was refined from 100 poses in MOE and CLUSPRO, revealing a preference for ceramide to bind ANXA2 on one face of the enzyme, interacting specifically with Glu142 (E142). (C) Skeletal model of ceramide interacting with residues of interest, particularly E142, on ANXA2. (D) Western blotting was performed to detect the protein abundance of stably knocked down ANXA2, and transiently transfected V5-tagged mutant or wildtype ANXA2. Actin was used as the loading control. (E–G) Uninfected UMSCC1A cells stably expressing shRNA against endogenous ANXA2 were transiently transfected with V5-wiltype ANXA2 or V5-E142A ANXA2 pcDNA3.1+ plasmid and treated with LCL768 (30 μM, 2 h) or vehicle control. Cells were dual labeled with ceramide (green) and V5 (red) antibody for confocal imagery. Similarly, ceramide (green) and LC3 (red), and LC3 (green) and Tom20 (red) confocal images are shown. Images represent three independent experiments. Yellow indicates colocalization. Quantification of colocalization in confocal images was estimated via Pearson’s correlation coefficient and normalized to exogenous wildtype ANXA2 expressing, untreated control cells. Values indicate mean ± SD of n = 3 independent experiments. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (H and I) UMSCC1A cells stably expressing shRNA against endogenous ANXA2 were transiently transfected with either V5-wildtype ANXA2 or V5-E142A ANXA2 pcDNA3.1+ plasmid, infected with WT P. gingivalis for 6 h (100 MOI), and treated with LCL768 (30 μM, 2 h) or vehicle control. Confocal microscopy was conducted on cells dual-labelled with FimA and V5-tag antibody. Quantification of colocalization via Pearson’s correlation coefficient was normalized to exogenous wildtype ANXA2 expressing, untreated, uninfected control cells. Values indicate mean ± SD of n = 3 independent experiments. Ns not significant, ∗∗ p < 0.01.

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Mutagenesis, Binding Assay, Western Blot, Quantitative Proteomics, Stable Transfection, Transfection, Control, Expressing, shRNA, Plasmid Preparation, Labeling, Infection, Confocal Microscopy

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: ANXA2 and LC3 knockdown in OSCC xenograft tumors resulted in attenuation of mitophagy (A) ANXA2, LC3, or scrambled control (SCR) shRNA were stably expressed in UMSCC1A cells. Cells were then injected into the midline flanks of nude mice and allowed to incubate for approximately 23 days before intraperitoneal treatment with LCL768/vehicle control (10 mg/kg, n = 8). Flank tumors were measured using calipers, and measurements indicate mean ± SD of n = 8 independent experiments. Student’s t test was conducted to examine significance in growth response curves (SCR VEH vs. LCL ∗∗ p < 0.01, ANXA2KD VEH vs. LCL Ns not significant, LC3KD vs. LCL Ns not significant). (B) Survival Kaplan-Meier curves of nude mice displaying xenograft tumors induced by injection with UM-SCC-1A OSCC cells stably expressing shRNA for ANXA2, LC3, or scrambled (SCR) control and treated/untreated with LCL768 ( n = 8 mice). ∗ p < 0.05. (C) Immunofluorescence was completed on sections of nude mouse xenograft flank tumors stably expressing either ANXA2, LC3, or scrambled (SCR) control shRNA and treated with LCL768 or vehicle control intraperitoneally (10 mg/kg). Quantification of confocal images of LC3 and Tom20, ceramide and LC3, Tom20 and LAMP1, and ceramide and ANXA2 association are shown. Pearson’s correlation coefficient values indicate mean ± SD of n = 3 independent experiments, normalized to uninfected, untreated control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (D) Mouse xenograft flank tumors injected with cells stably expressing ANXA2, LC3, or SCR shRNA and treated/untreated with LCL768 were stained with Ki67 antibody, indicating proliferating tumor cells. (E) Quantification of Ki67+ nuclei staining in tumor sections was performed in inform Automated Image Analysis software and represents a mean ± SD of n = 3 independent experiments.

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Knockdown, Control, shRNA, Stable Transfection, Injection, Expressing, Immunofluorescence, Staining, Software

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet: Orthotopic mouse oral squamous cell carcinoma tumors exhibit inhibited mitophagy when orally infected with P gingivalis (A) Orthotopic oral squamous cell carcinoma tumors were generated in female C57BL/6 mice (6–8 weeks old) by injecting MOC2 cells intramuscularly into the mylohyoid. Tumors developed over at least 2.5 weeks before uninfected (NP) mice were treated with 10 mg/kg LCL768 intraperitoneally every other day for 2.5 weeks. Otherwise, the resident mouse oral microbiota was treated with antibiotic for 3 days in drinking water prior to starting concurrent P. gingivalis infection (10 9 CFU, oral gavage) and LCL768/vehicle intraperitoneal treatment every other day. P. gingivalis infection via oral gavage was performed four times total. (B) Fluorescent in situ hybridization (FISH) was conducted on mouse OSCC tumor sections infected/uninfected with P. gingivalis and treated with LCL768 or vehicle control to confirm the presence of the bacterium in tumor samples. Insets (right panels) show higher magnifications marked with white squares in the middle panels. (C) Quantification of P. gingivalis signal foci mean fluorescence intensity in (B) was completed with ImageJ Fiji and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. Ns, not significant, ∗∗∗∗ p < 0.0001. (D–G) Immunofluorescence was completed on sections of female C57BL/6 mouse orthotopic oral squamous carcinoma tumor infected or uninfected with P. gingivalis (10 9 CFU) via oral feeding and treated with LCL768 or vehicle control intraperitoneally (10 mg/kg). Quantification of confocal images of LC3 and Tom20 (D), ceramide and LC3 (E), Tom20 and LAMP1 (F), ANXA2 and ceramide (G) association are shown. Pearson’s correlation coefficient values indicate mean ± SD of n = 3 independent experiments, normalized to uninfected, untreated control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (H) Mouse OSCC tumors were stained with Ki67 antibody, indicating proliferating tumor cells. (I) Quantification of Ki67+ nuclei staining in mouse orthotopic tumor sections in (H). (J) Basal percentage of tumor infiltrating lymphocytes in mouse orthotopic tumors.

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Infection, Generated, In Situ Hybridization, Control, Fluorescence, Immunofluorescence, Staining

Journal: iScience

Article Title: Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

doi: 10.1016/j.isci.2024.109860

Figure Lengend Snippet:

Article Snippet: Five Micrograms (5 μg) of recombinant human ANXA2 (R&D Systems, #9409-AN-050) was resolved by SDS-PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad, #4568094) as previously described in Far Western methodologies., The SDS-PAGE resolved protein samples were then transferred to a 0.22 μm PVDF membrane and were blocked overnight in 5% non-fat milk.

Techniques: Virus, Mutagenesis, Recombinant, MTT Cell Proliferation, Transfection, Lysis, Protease Inhibitor, Membrane, Silver Staining, In Situ Hybridization, Plasmid Preparation, In Situ, Isolation, Cell Culture, Western Blot, shRNA, Expressing, Software

Journal: PLoS ONE

Article Title: Anxa2 attenuates osteoblast growth and is associated with hip BMD and osteoporotic fracture in Chinese elderly

doi: 10.1371/journal.pone.0194781

Figure Lengend Snippet: Presented are Anxa2 protein abundances (Label-Free Quantification [LFQ] intensity) in PBM in Sample 1. The data is described as mean±SD. **: P<0.01, as compared with NF subjects.

Article Snippet: The recombinant protein of human Annexin A2 (Origene, Catalogue No.TP305081) was then added to the medium generating various concentrations (0, 25, 50, 100, 200ng/ml).

Techniques: Quantitative Proteomics

Journal: PLoS ONE

Article Title: Anxa2 attenuates osteoblast growth and is associated with hip BMD and osteoporotic fracture in Chinese elderly

doi: 10.1371/journal.pone.0194781

Figure Lengend Snippet: The correlation between Anxa2 protein level in plasma and hip BMD was analyzed with Pearson correlation analysis (r = -0.302, P = 0.0084).

Article Snippet: The recombinant protein of human Annexin A2 (Origene, Catalogue No.TP305081) was then added to the medium generating various concentrations (0, 25, 50, 100, 200ng/ml).

Techniques: Clinical Proteomics

Journal: PLoS ONE

Article Title: Anxa2 attenuates osteoblast growth and is associated with hip BMD and osteoporotic fracture in Chinese elderly

doi: 10.1371/journal.pone.0194781

Figure Lengend Snippet: Presented are Anxa2 protein abundances in plasma in Sample 2. The data is described as mean±SD. *: P<0.05, as compared with high BMD subjects.

Article Snippet: The recombinant protein of human Annexin A2 (Origene, Catalogue No.TP305081) was then added to the medium generating various concentrations (0, 25, 50, 100, 200ng/ml).

Techniques: Clinical Proteomics

Journal: PLoS ONE

Article Title: Anxa2 attenuates osteoblast growth and is associated with hip BMD and osteoporotic fracture in Chinese elderly

doi: 10.1371/journal.pone.0194781

Figure Lengend Snippet: Presented are cell indexes (CI) monitored for 36 hours in cell growth assay. The data is described as mean±SD. It reflected the inverse U-shaped effect of Anxa2 protein on hFOB growth at each representative time point (P = 0.0064).

Article Snippet: The recombinant protein of human Annexin A2 (Origene, Catalogue No.TP305081) was then added to the medium generating various concentrations (0, 25, 50, 100, 200ng/ml).

Techniques: Growth Assay