Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Autophagic Lifestyle of P. gingivalis (P. g) is Highly Characterized by Only the LC3C Isoform of LC3, Which is not Increased During Starvation-Induced Autophagy in GECs. (A ) The LC3 A/B lipidation results of the same assay provided in . (B ) GECs were separately treated with LC3B siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6 h. Intracellular P. g survival after LC3B siRNA depletion was determined using a standard antibiotic protection assay using P. g- specific 16S rRNA primers. ( C ) GECs were subjected to starvation conditions in HBSS for 24 h. GECs were then collected and fixed so that immunofluorescence could be performed. GECs were stained for LC3C (rabbit anti-LC3C;Alexa 568; red). GECs were then imaged via confocal microscopy (Super Resolution Zeiss Airyscan LSM 880) at 63x. Western blotting (Not Shown) was utilized to confirm the lack of induction of LC3C I and LC3C II. Data is represented as Mean±SD; n=3; p<0.05 is considered statistically significant (Student two-tailed T-test).

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Causes the Nucleation of Hsp27-Mediated LC3C Accumulation and Lipidation; this Specific Assembly is Highly Dependent on Host Cells’ Redox Potential Determined by eATP Treatments. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated 6 and 24 h. Non-Depleted and HSp27-depleted GECs also were treated with the physiologically-relevant oxidative stress inducer eATP (3mM) treatment for 30 min prior to infection, and were analyzed by western blot.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Infection, Western Blot

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis ( P. g ) Induces and Prolongs the Autophagosomal LC3C/Beclin 1/ATG14 Nucleation Complex in a Manner Dependent upon HSp27 and the Reduced Redox State of Infected GECs as Determined by Isolated P. g- Specific Autophagosomes. GECs were treated with HSP27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC)(50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 and 12 h. Autophagosomes were then isolated and prepared for analysis. ( A ) The glutathione (GSH) levels of primary GECs were also measured using chemiluminescence detection. ( B) Isolated autophagosomes were analyzed via western blot. ( Bi ), ( Bii ), ( Biii ) Quantitative ImageJ analysis was performed of each of the western blot results. Data is represented as Mean±SD, where n=3 for results. p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Isolation, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Recruit Beclin 1 and ATG14 to Form a Temporal Pro-bacterial Autophagic Complex, which Can Be Disrupted by Increased Oxidative Stress. Human Primary GECs were treated with HSp27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. gingivalis (P. g) was added at MOI 100 to GECs, which were incubated 6 and 12 h. GECs were then lysed and the extracts were incubated in rabbit anti-LC3C antibody over-night. Samples underwent co-immunoprecipitation. ( A ) The eluted protein complexes were then analyzed by western blot. ( Ai ), ( Aii ), and ( Aiii ) Quantitative ImageJ analysis of western blot results was performed for each of the proteins in question. ( B ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. The Imaris software was used to obtain zoomed orthogonal views of ( Bi ) An infected GECs and ( Bii ) a theoretical autophagosome with HSp27, LC3C, ATG14, and Beclin 1 highly co-localized about it. The scale bar is 20 µm for all Magnification. All of the markers were found to have a Pearson correlation coefficient greater than .9 with each other via Imaris, denoting their close theorized interactions. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Immunoprecipitation, Western Blot, Staining, Software, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Selectively and Specifically Partner with One Another to Promote P. gingivalis ( P. g )-Induced Autophagy. P. g was added at MOI 100 to Human Primary GECs, which were incubated 6 and 24 h. ( A ) GECs were stained for LC3C (rabbit anti-LC3C; Alexa 488; green) and HSp27 (mouse anti-HSp37; Alexa 568; red) following infection. HSp27 was found to readily and temporally colocalize with LC3C, having a Pearsons correlation coefficient of .85 at 24 h post infection via the Imaris post-processing software. ( B ) To assess if full length HSp27 is truly capable of binding to full-length LC3C, a far western approach was implemented by probing 5 µg of recombinant LC3C with 10 µg of recombinant HSp27 for one hour. Antibody specificity was accounted for via probing the LC3C blot with monoclonal mouse anti-HSp27 antibody (Not shown), which showed no cross-reactivity.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Staining, Infection, Software, Binding Assay, Western Blot, Recombinant

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 does not Interact with either LC3A or LC3B isoforms in the way that it interacts with LC3C. A Far Western approach was implemented. rLC3A or rLC3B were loaded and incubated with 10 ug of rHSp27. Interactions for ( Bi ) LC3A and ( Bii ) LC3B were then detected by probing the rLC3A or rLC3B blot with mouse Anti-Hsp27 antibody.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Phosphorylated HSp27 (P-HSp27) Preferentially Binds to the C-terminal Tail of LC3C, Inhibiting the Canonical Cleavage of LC3C and Halting the Canonical Maturation of LC3C-Specific Autophagosomes. The structural models of monomeric full-length wild-type ( A ) HSp27 (Uniprot: P04792) and ( B ) LC3C (Uniprot: Q9BXW4) were acquired from the AlphaFold database. Optimized complex configurations between ( C ) LC3C and unmodified HSp27 and ( D ) LC3C and P-HSp27 were then obtained, and the modifications in the theorized interaction sites in their N-terminal regions were highlighted. ( E ) Surface electrostatic potentials of the complexes were additionally mapped, contrasting the varied potentials between the two complexes. ( F ) Finally, the buried surface areas and interaction areas between HSp27 or P-HSp27 and LC3C proteins were also assessed and contact maps were generated.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Generated

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Secretes its Ndk Effector Molecule to Activate HSp27 and Induce Temporal HSp27-LC3C Partnering to Inhibit Canonical LC3C Cleavage by ATG4B and Halt Autolyosomal Fusion in GECs. A) Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h or were transfected with 1 µg of the constitutively activated pFLAG-CMV2-HSP27-S78D/S82D construct for 48h. Select GECs were then jointly treated with the late stage autophagy inhibitors 1 µM Pepstatin A, or 1 µM lactostatin for 24h. Wild-type P. g or ΔNDK P. g was then added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( B ) A diagram was created detailing how LC3C preferentially partners to P-HSp27 over its non-phosphorylated counterpart, causing a confirmational shift to the C-terminal tail of LC3C. This shift results in the inhibition of the final lipidated LC3C tail cleavage by the ATG4B protease, lending to LC3C not disassociating from the autophagosome and halting fusion with the lysosome. ( C ) A diagram was also created to highlight the temporal relationship between HSp27 and LC3C, where LC3C can initially bind to HSp27 but via the actions of Ndk, it preferentially binds to P-HSp27, resulting in a limiting of mature, cleaved LC3C.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Construct, Incubation, Staining, Inhibition

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Quantifications of Cross-Sectional Human Ex-Vivo Samples Support High Levels of P. gingivalis (P. g) , HSp27, and LC3C in Chronically Diseased Oral Tissues (i.e. Periodontitis). Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis patients were obtained using the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System so that the mean fluorescence intensity of ( A ) HSp27 ( B ) P. g and ( C ) LC3C could be calculated using ImageJ with JACoP Plugin. Data are presented as mean ± SD. Representative images from at least 5 different patients per group were used for quantitative analysis and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Ex Vivo, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 is a Critical Regulator in Pro-bacterial LC3C-Characterized Autophagy, Facilitating the Intracellular Autophagic Survival of P. gingivalis (P. g) and Influencing the Bacterial Symbiosis of the Oral Mucosa. The proposed diagram of the identified mechanisms of P. gingivalis persistence in GECs. ( A ) HSp27 is largely induced and spatially recruited by P. g invasion of the host cells. After the initial periods of cellular infection, the gradually growing secretion of the bacterial Nucleoside-diphosphate-kinase (Ndk) into the cytoplasmic space causes heightened activation of HSp27 (P-HSp27) via direct phosphorylation. P-HSp27 abrogates extracellular ATP (eATP)-induced antimicrobial Reactive-Oxygen-Species (ROS) production via increasing glutathione (GSH) levels. In parallel, P. g -mediated induction of HSp27 promotes the specific recruitment and lipidation of LC3C, an isomer of the LC3 autophagosomal structural molecule, which is strictly dependent upon the large presence and the strong antioxidant activity of HSp27. LC3C and HSp27 partner in a stepwise manner, 1) their coupling drives the formation of Beclin1/ATG14 induction, 2) where the temporally increased phosphorylation of HSp27 by P. g Ndk both strengthens the P-HSP27 and LC3C partnering and shifts the confirmation of the LC3C tail so that LC3C cannot be successfully further cleaved by ATG4. ( B ) P-HSp27 and LC3C become increasingly assembled to the ATG14-Incorperated Nucleation Complex, where they prolong the complex’s formation and result in the accumulation of the complex on forming autophagic membranes. Thus, the strengthened partnering between P-HSp27 and LC3C is the proposed mechanism for inhibiting the autolysosomal fusion of P. g- specific autophagosomes, which is also controlled by the host cell redox homeostasis ( C ) Autophagic P. g does not undergo lysosomal degradation and is instead able to survive, multiply and subsequently intercellularly spread to neighboring cells to propagate. ( D ) Thus, the non-canonical, pro-bacterial autophagic events create a favorable and protected cellular environment for P. g , thereby establishing long-term intracellular bacterial persistence. The chronic colonization of P. g in the epithelia can lead to host-microbial dysbiosis in oral mucosa and systemic disorders.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Activation Assay, Phospho-proteomics, Antioxidant Activity Assay

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: CALCOCO1 interacts with MAP1LC3C and other LC3/GABARAP family members. (A) Cartoon of domains and motifs found in human and mouse CALCOCO family members. (B) Immunoprecipitation (IP) analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, resolved by SDS-PAGE, and probed with anti-HA or anti-DDK antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (C) IP analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs expressed in HEK293 cells that were treated overnight with chloroquine (CQ) to inhibit lysosomal flux. HA-tagged protein complexes were IP’d with anti-HA antibody and probed with anti-DDK or mixed antibodies against HA, MAP1LC3A/B, and MAP1LC3C. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (D) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with wild type (WT) CALCOCO1-MYCDDK, CLIR mutants L140A and V142A, and LIR mutant W47A in HEK293 cells. CALCOCO1W47A was transfected at 2 different concentrations to match WT expression levels. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, and probed with anti-HA or anti-CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as noted. (E) IP analyses of CALCOCO1-MYCDDK with endogenous MAP1LC3B. WT CALCOCO1-MYCDDK, 2 concentrations of the CALCOCO1W47A, and GFP-DDK (control) were expressed in HEK293 cells, then treated for 18 h with 100 nM MLN0128 and 100 uM CQ. CALCOCO1-MYCDDK complexes were immunoprecipitated with anti-Flag® M2 affinity gel (MilliporeSigma, A2220) and probed with anti-DDK or MAP1LC3B antibodies. Expressions of CALCOCO1-MYCDDK, GFP-DDK, ACTB and MAP1LC3B in cell lysates are shown with antibodies as noted. (F) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with WT CALCOCO1-MYCDDK, CLIR mutants (L140A and V142A), and R12H mutant in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody and probed with anti-HA or anti-CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies, as noted

Article Snippet: Antibodies Primary antibodies used in this study include: Abcam antibodies for CALCOCO1/CoCoA (ab70564); ABD SeroTech antibodies GAPDH (HCA272); BD Biosciences antibodies for SQSTM1 (610832) and RPS6KB1 (611261); Cell Signaling Technology antibodies for ATG3 (3415), ATG5 (8540), ATG7 (8558), MAP1LC3A/B (12741), MAP1LC3C (14723), ULK1 (6439 and 8054), p-EIF4EBP1 T37/46 (2855), p-EIF4EBP1 S65 (9456), EIF4EBP1 (9644) p-AKT1 S473 (4060), AKT1 (9272) p-RPS6KB T389 (9234), p-ACACA S79 (11818), ACACA (3662) DYKDDDDK-tag (14793), MYC-tag (2276), ACTB (beta-Actin; 3700), LMNA (lamin A/C; 4777); Novus antibodies for MAP1LC3B (NB100-2220); Protein Tech antibodies for RETREG1 (21537-1-AP), GABARAPL2 (18724-1-AP), MAP1LC3C (18726-1-AP); Santa Cruz Biotechnology antibodies for CALCOCO1 (sc-515670), HA-tag (sc-805), GST-tag (sc-138), TUBA (alpha-tubulin; sc-5286); MilliporeSigma antibody for Flag M2 (F1804).

Techniques: Immunoprecipitation, SDS Page, Mutagenesis, Transfection, Expressing

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Myc, GFP, or 3xFlag tag is fused to human MAP1LC3B ORF at its N- terminus. The box bars on the left represent the N-terminal tags. The dashed lines represent MAP1LC3B with 27 amino acids sequence shown at the carboxyl terminus. The arrow points to Glycine 120 conjugation site. The last five amino acids at the C-terminal are underlined. Mutant amino acid or last amino acid of LC3B is bolded. Human LC3C, Rat LC3, Rat LC3B, Mouse LC3A, Mouse LC3B and the related mutants are described in Result section.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Sequencing, Conjugation Assay, Mutagenesis

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Pairwise alignments of LC3A, LC3B and LC3C of human and rodent origin are generated by ClustalW2. The glycine conjugation sites are boxed and C-terminal amino acids after glycine conjugation site are underlined. A “*” presents identity of amino acids; “ : ” presents these amino acids in a same amino acid group (similarity); “⋅” presents different group of amino acids.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Generated, Conjugation Assay

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, Compare pro-human LC3B with pro-mouse LC3B, pro-rat LC3B and their cleaved and lipidated forms’ band patterns by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. B, compare pro-rat LC3B with pro-rat LC3 and cleaved forms by 15% of SDS-polyacrylamide gel. Correspondent expression plasmids were expressed in HEK293 cells. Note: human LC3B-I band is close to RatLC3B-I/RatLC3-I as indicated by arrows. C, pro-LC3A and its cleaved form comparison by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. D, compare pro-human LC3B and LC3C species by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells for 24 h. HEK293 cells transient expressing Human LC3C were treated with 50 µM of CQ for 2 h to induce accumulation of human LC3B-II and LC3C-II. Total cell lysates were separated by a 15% of SDS-polyacrylamide gel. Protein transferred membrane was cut between human LC3B group and human LC3C group and immunoblotting by LC3B and LC3C antibody, respectively. The immunoblotting membrane was re-aligned before developing with chemiluminescent substrate in order to compare human LC3B species and human LC3C species in Wb.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Comparison, Membrane, Western Blot

Journal: Frontiers in Pharmacology

Article Title: SC75741, A Novel c-Abl Inhibitor, Promotes the Clearance of TDP25 Aggregates via ATG5-Dependent Autophagy Pathway

doi: 10.3389/fphar.2021.741219

Figure Lengend Snippet: SC75741-induced TDP25 degradation depends on p62 and LC3C. (A) . HEK293 WT and p62-/- cells were transfected with GFP-TDP25 for 24 h, treated with SC75741 (5 μM) for 12 and 24 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (B) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (C) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 8, **** p < 0.0001, two-tailed t test). (D) . HEK293 WT cells were transfected with GFP-TDP25 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction between p62 and TDP25 was analyzed by immunoprecipitation. (E) . Hela WT and ATG8-/- cells were transfected with GFP-TDP25 for 24 , treated with SC75741 (5 μM) for 3 and 6 . Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (F) .Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (G) . Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 10, **** p < 0.0001, two-tailed t test). (H) . HEK293 p62-/- cells were transfected with GFP, GFP-TDP25, Flag and Flag-p62 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction of p62 with TDP25 and LC3C were analyzed by immunoprecipitation. (I) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6 h and the co-localization among TDP25, p62, lamp1 were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm. (J) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6h and the co-localization among TDP25, p62, LC3C were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm.

Article Snippet: The following antibodies were used in this study: β-tubulin (#M1305-2), Flag-Tag (#M1403-2), HA-Tag (#0906-1), GFP-Tag (#ET1607-31) from HuaAn Biotechnology; c-Abl (#2862), phospho-c-Abl (Tyr245) (#2861), TFEB (#37785), NF-κB (#8242), phospho-NF-κB (#5733) from Cell Signaling Technology®; LC3C (#18726-1-AP), p62 (#18420-1-AP) from Proteintech TM; LC3C (#A8295), Lamp1 (#A16894), TFEB (#A7311) from ABclonal; The secondary antibodies for western blotting were used: Goat anti-Mouse IgG (H + L) (#31430, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H + L) (#31460, Thermo Fisher Scientific); The fluorescent secondary antibodies for immunofluorescence were used: goat anti-rabbit Alexa Fluor 546 (#A-11010), goat anti-mouse Alexa Fluor 405 (#A-31553) from Thermo Fisher Scientific.

Techniques: Transfection, Fluorescence, Microscopy, Two Tailed Test, Immunoprecipitation, Immunofluorescence, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: SC75741, A Novel c-Abl Inhibitor, Promotes the Clearance of TDP25 Aggregates via ATG5-Dependent Autophagy Pathway

doi: 10.3389/fphar.2021.741219

Figure Lengend Snippet: SC75741-induced TDP25 degradation depends on p62 and LC3C. (A) . HEK293 WT and p62-/- cells were transfected with GFP-TDP25 for 24 h, treated with SC75741 (5 μM) for 12 and 24 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (B) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (C) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 8, **** p < 0.0001, two-tailed t test). (D) . HEK293 WT cells were transfected with GFP-TDP25 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction between p62 and TDP25 was analyzed by immunoprecipitation. (E) . Hela WT and ATG8-/- cells were transfected with GFP-TDP25 for 24 , treated with SC75741 (5 μM) for 3 and 6 . Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (F) .Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (G) . Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 10, **** p < 0.0001, two-tailed t test). (H) . HEK293 p62-/- cells were transfected with GFP, GFP-TDP25, Flag and Flag-p62 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction of p62 with TDP25 and LC3C were analyzed by immunoprecipitation. (I) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6 h and the co-localization among TDP25, p62, lamp1 were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm. (J) . HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6h and the co-localization among TDP25, p62, LC3C were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm.

Article Snippet: The following antibodies were used in this study: β-tubulin (#M1305-2), Flag-Tag (#M1403-2), HA-Tag (#0906-1), GFP-Tag (#ET1607-31) from HuaAn Biotechnology; c-Abl (#2862), phospho-c-Abl (Tyr245) (#2861), TFEB (#37785), NF-κB (#8242), phospho-NF-κB (#5733) from Cell Signaling Technology®; LC3C (#18726-1-AP), p62 (#18420-1-AP) from Proteintech TM; LC3C (#A8295), Lamp1 (#A16894), TFEB (#A7311) from ABclonal; The secondary antibodies for western blotting were used: Goat anti-Mouse IgG (H + L) (#31430, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H + L) (#31460, Thermo Fisher Scientific); The fluorescent secondary antibodies for immunofluorescence were used: goat anti-rabbit Alexa Fluor 546 (#A-11010), goat anti-mouse Alexa Fluor 405 (#A-31553) from Thermo Fisher Scientific.

Techniques: Transfection, Fluorescence, Microscopy, Two Tailed Test, Immunoprecipitation, Immunofluorescence, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: SC75741, A Novel c-Abl Inhibitor, Promotes the Clearance of TDP25 Aggregates via ATG5-Dependent Autophagy Pathway

doi: 10.3389/fphar.2021.741219

Figure Lengend Snippet: SC75741 activates TFEB independently of mTORC1 activity. (A) . H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, imatinib (5 μM) or SC75741 (5 μM) for another 24 h. Nuclear lysates and cytoplasmic lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin, TFEB/H3). (B) . H4GT25 cells were pretreated with 1 µg/mL DOX for 24h, treated with SC75741 (5 μM) for 12 and 24 h. Nuclear lysates and cytoplasmic lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin, TFEB/H3). (C) . H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, imatinib (5 μM) or SC75741 (5 μM) for another 12h, and then the cells were subjected to immuno-fluorescence analysis using an anti-TFEB antibody. Scale bar, 40 μm. (D) . Quantification of the percentage of TFEB-positive cells in the nucleus and TFEB-positive cells in the cytoplasm. (data represents mean ± SD; DMSO ( n =110 cells), imatinib ( n =112 cells), SC75741 ( n =101 cells)). (E) . H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, Torin1 (1 μM), imatinib (5 μM) or SC75741 (5 μM) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin). (F) . q-PCR analysis of different mRNA TFEB target genes in H4GT25 cells treated with imatinib (5 μM) and SC75741 (5 μM) for 12 h. (G) . H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, Torin1 (1 μM), imatinib (5 μM) or SC75741 (5 μM) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (p-S6K/Tubulin, S6K/Tubulin,).

Article Snippet: The following antibodies were used in this study: β-tubulin (#M1305-2), Flag-Tag (#M1403-2), HA-Tag (#0906-1), GFP-Tag (#ET1607-31) from HuaAn Biotechnology; c-Abl (#2862), phospho-c-Abl (Tyr245) (#2861), TFEB (#37785), NF-κB (#8242), phospho-NF-κB (#5733) from Cell Signaling Technology®; LC3C (#18726-1-AP), p62 (#18420-1-AP) from Proteintech TM; LC3C (#A8295), Lamp1 (#A16894), TFEB (#A7311) from ABclonal; The secondary antibodies for western blotting were used: Goat anti-Mouse IgG (H + L) (#31430, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H + L) (#31460, Thermo Fisher Scientific); The fluorescent secondary antibodies for immunofluorescence were used: goat anti-rabbit Alexa Fluor 546 (#A-11010), goat anti-mouse Alexa Fluor 405 (#A-31553) from Thermo Fisher Scientific.

Techniques: Activity Assay, Fluorescence