Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: Downregulation of BAG3 affects p-STAT3(705) and p-STAT3(727)

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 regulates p-STAT3(705) and p-STAT3(727) through p-JAK2 and p-ERK1/2 respectively to promote VSMCs migration

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques: Migration

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 modulates STAT3 phosphorylation by regulating the interaction of STAT3 with JAK2 and ERK1/2

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Nature Communications

Article Title: Bipolar and schizophrenia risk gene AKAP11 encodes an autophagy receptor coupling the regulation of PKA kinase network homeostasis to synaptic transmission

doi: 10.1038/s41467-025-66356-w

Figure Lengend Snippet: a The predicted kinase activity scores for representative kinases. The scores were generated by PhosR based on the phosphorylation levels of predicted substrates. Statistical significance was determined using two-sided t -test. b, d Volcano plot of the top 200 predicted GSK3β ( b ) / AKT1 ( d ) phosphorylation sites (normalized by the total protein level) detected by mass-spectrometry in Akap11 -cKO mouse brains. A positive score indicates enrichment, a negative score indicates depletion. The y axis represents statistical confidence for each x axis point. Enriched proteins, defined by FDR < 0.05 and |Log2FC | > 2 SD, are colored in red. FDR was calculated with the moderated two-sided t -test in the LIMMA package for multiple comparisons. c Immunoblotting using antibodies against AKAP11, GSK3α/β and p-GSK3α/β (S21/S9) and quantification of the results. Data are presented as mean ± SEM ( n = 11 per genotype). Statistical analysis was performed using an unpaired two-tailed t -test (p-GSK3α/GSK3α, p = 0.0036; p-GSK3β/ GSK3β, p = 0.0053). Each experiment was independently repeated three times with similar results. e Immunoblotting using antibodies against p-GSK3α/β (S21/S9), GSK3α/β, p-Akt1 S473 , and Akt1 in cytosolic and synaptosome fractions and quantification of the results. Data are presented as mean ± SEM (p-GSK3β: n = 4 per genotype; p-Akt1: n = 5 for Akap11 F /F and n = 4 for Akap11 F/F ; Syn-cre). Statistical analysis was performed using an unpaired two-tested t -test (cytosolic p-GSK3β: p = 0.0007; synaptosome p-GSK3β: p = 0.1206; synaptosome p-Akt1: p = 0.0154). Each experiment was independently repeated three times with similar results.

Article Snippet: Primary antibodies, Rabbit anti-AKAP11 (LS-Bio, LS-C374339), Rabbit anti-RIα (Cell signaling, 5675S), Sheep anti-RIβ (R&D, AF4177), Mouse anti-RIIα (BD, 612242), Rabbit anti-PKACα (Cell signaling, 4782S), Rabbit anti-p-PKACα T197 (Cell signaling, 5661S), Mouse anti-β-actin (Cell signaling, 3700S), Rabbit anti-ADRA2A (Proteintech, 14266-1-AP), Rabbit anti-SPHKAP (Thermo, PA5-27581), Rabbit anti-Synaptophysin (Thermo, MA5-14532), Rabbit anti-GSK3α/β (Cell signaling, 5676S), Rabbit anti-p-GSK3α/β (S21/S9) (Cell signaling, 9331S), Rabbit anti-p-GluA1 S845 (Cell signaling, 8084), Rabbit anti-GluA1 (Cell signaling, 13185), Rabbit anti-Akt1 (Cell signaling, 4691S), Rabbit anti-p-Akt1 S473 (Cell signaling, 4060S), Chicken anti-GFP (Invitrogen, A10262), Rabbit anti-VAPA (Proteintech, 15275-1-AP), Rabbit anti-VAPB (Proteintech, 14477-1-AP), Guinea pig anti-p62 (PROGEN, GP62-C), Rabbit anti-LC3A/B (Cell signaling, 12742S), were diluted in blocking buffer with 0.2% Tween-20 and incubated overnight at 4 °C.

Techniques: Activity Assay, Generated, Phospho-proteomics, Mass Spectrometry, 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: P. gingivalis (P. g) Infection Causes a Significant Increase in the Protein HSp27, Accompanied by Large Spatial Accumulation of Hsp27 with the Bacteria in a Temporal Manner in Primary GECs. ( A ) Representative confocal microscopy images of P. g -infected human primary GECs at an MOI 100, at 6 h and 24 h after infection. GECs were then stained for P. g (rabbit anti-P. g; Alexa 488; green) or HSp27 (mouse anti-HSp27; Alexa 568; red). GECs were then imaged via the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( Ai ) Imaris Software was used to create a zoomed image of infected GECs and was used to calculate the amount of co-localization between P. g and HSp27. HSp27 was found to readily colocalize with P. g , having an average Pearson correlation coefficient of 0.87 via the Imaris software. Scale bar is 30 µm for 63x and Zoomed Magnification. (B ) P. g was added at MOI 100 to GECs, which were incubated 6 or 12h. Cell lysates were then analyzed via western blot. ( Bi ) Quantitative ImageJ analyses of western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as significant via Two-Tailed Student T-test. *p<0.05.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Bacteria, Confocal Microscopy, Staining, Software, 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: The Integrity of P. gingivalis (P. g) -Specific Autophagosomes is Highly Dependent on HSp27 Presence. Human primary GECs were treated with HSP27siRNA (100nM) for 48 h prior to incubation with P. g ( MOI 100) for 6 h. Autophagosomes were then isolated and analyzed via Confocal Microscopy. ( A ) Schematic autophagosomal isolation method of infected GECs. ( B ) Confocal microscopy images of autophagosomes (ThiolTracker Violet; blue) were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( C ) Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal microscopy images of autophagosomes were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( Ci ) Quantitative ImageJ analysis of Confocal microscopy results was then performed. Data is represented as Mean±SD, where n=25 and p<0.05 was considered as statistically significant (Student two-tailed T-test). **p<.005

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Isolation, Confocal Microscopy, Infection, Staining, 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 Magnetic-Labelling Process Does Not Impact P. gingivalis (P. g) Infection in GECs. P. g was labeled with lipobiotin (5 μM) and were then incubated in MagCellect Streptavidin Ferrofluid. Human Primary GECs were then infected for 24 h. Representative confocal microscopy images of P. g- infected GECs at an MOI 100, were taken at 24 h after infection using via Zeiss LSM 880 (63x). P. g (rabbit anti-P . gingivalis ; Alexa 488; green) was detected in the GECs. Actin (red) was stained utilizing Rho-Phallodin.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Incubation, Confocal Microscopy, Staining

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: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 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: 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: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 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: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 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: Δ ndk-P. gingivalis (P. g) Undergoes Canonical Degradative Autophagosomal Trafficking in Human Primary GECs. Primary GECs were infected with WT P. gingivalis (P. g) versus Δ ndk - P. g at MOI 100 for 3, 12, or 24h. ( A ) TEM analysis used Immunogold labeling for P. g was performed on WT P. g versus Δ ndk - P. g at 3 or 24h post-infection. Images were acquired at 40000x magnification utilizing a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc.) affixed to a Veleta camera with iTEM. ( 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. Co-localization analysis of P. g and LAMP-1 was additionally carried out using the Zeiss LSM 880 Confocal Software, determining that the WT P. g had a Pearsons value of .137 while the Δ ndk - P. g had a Pearsons value of .704.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Staining, Software

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 Depletion of HSp27 Abrogates the Inhibition of Oxidative Stress and Severely Impacts the Intracellular Survival of P. gingivalis (P. g) Studied in Human Primary Organotypic Cultures of Gingiva. To create the organotypic culture systems, human primary GECs and Fibroblasts Cells (FBCs) were co-cultured together upon a collagen raft. Select rafts were then treated with HSP27 siRNA (100nM) for 48 h. Select rafts were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h. P. g was added at MOI 100 to rafts, which were incubated for 24 h. Rafts were then collected and sectioned so that immunofluorescence could be performed. ( A ) Representative images of H&E stained raft culture systems at 20x magnification, which clearly mimic the oral gingival crevice. Scale bar is 50 µm. E: Multilayer Undifferentiated Epithelial Cells, C: Collagen Matrix; F: Fibroblasts. ( B ) Rafts were stained for P. g (rabbit anti- P. g; Alexa 488; green) or HpS27 (mouse anti-HSp27; Alexa 568; red). Rafts were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 20x and 63x. Scale bar is 50 um for 20x and 20 um for 63x and Zoomed View. The range of z-stacks was kept consistent. HSp27 was once again found to readily co-localize with P.g with a Pearson coefficient of .83 as determined via the Imaris Software. ( Bi ) A Zoomed (4x) version of the 63x magnification was created using the Imaris Software, highlighting the intracellular nature of individual P. g. ( C ) GECs were additionally treated with HSp27 siRNA (100nM) for 48 h. Select GECs were then 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 h. If any extracellular bacteria were present, they were killed by gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment for 1 h. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular level of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via One-Way Anova Test. ** p<0.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Inhibition, Cell Culture, Incubation, Immunofluorescence, Staining, Confocal Microscopy, Software, Bacteria, Synthesized

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: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

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