Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Primers used for QPCR validation of microarray data

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Biomarker Discovery, Microarray

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Regulation of Klf isoform expression by ET-1 in cardiac myocytes (microarray analysis)

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Expressing, Microarray

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Regulation of Klf expression by ET-1. Cardiac myocytes were exposed to 100 nM ET-1 for the times indicated. RNA was extracted and expression of mRNAs for different Klf family members (A, Klf2; B, Klf4; C, Klf6; D, Klf5; E, Klf9; F, Klf10; G, Klf3; H, Klf11; I, Klf15) analysed by qPCR. Results are expressed relative to unstimulated controls and are means ± S.E.M. for at least 4 independent preparations of myocytes.

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Expressing

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Klf2, Klf4 and Klf6, but not Klf5, are upregulated as immediate early genes by ET-1. Cardiac myocytes were unstimulated or exposed to 20 μM cycloheximide (CX, open bars), 100 nM ET-1 (black bars), or ET-1 in the presence of cycloheximide (grey bars) for 0.5 h (Klf2, Klf4, Klf6) or 1 h (Irs2, Klf5, Il1rl1). RNA was extracted and expression of mRNAs for Klf2, Klf4, Klf6 and Irs2 (A, immediate early genes) or Klf5 and Il1rl1 (B, second phase genes) analysed by qPCR. Results are expressed relative to unstimulated controls and are means ± S.E.M. for 3 or 4 independent preparations of myocytes. ⁎ p < 0.01 relative to ET-1 alone (one-way ANOVA repeated measures with TUKEY post-test).

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Expressing

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Upregulation of Klf2, Klf4, Klf6 and Klf5 by ET-1 is mediated in part through the ERK1/2 cascade. Cardiac myocytes were unstimulated (Control), or exposed to inhibitors alone (10 μM U0126, 50 μM LY294002, 5 μM SB203580), to 100 nM ET-1 alone or to ET-1 in the presence of each inhibitor for 0.5 or 1 h. RNA was extracted and expression of mRNAs for Klf2, Klf4, Klf6 or Klf5 analysed by qPCR. A, Effects of U0126 on expression of Klf mRNAs at 0.5 (Klf2) or 1 h (Klf4, Klf5, Klf6). B, Effects of LY294002 or SB203580 on expression of Klf mRNAs at 1 h. Results are expressed relative to unstimulated controls and are means ± S.E.M. for 4 or 5 independent preparations of myocytes. ⁎ p < 0.05, # p < 0.001 relative to ET-1 alone (one-way ANOVA repeated measures with TUKEY post-test).

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Control, Expressing

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: Translation-state analysis of Klf mRNAs. Cardiac myocytes were unstimulated (Control) or exposed to 100 nM ET-1 (1 h). RNA was extracted for the total RNA pool. Polysomes were prepared from the same myocyte preparations using sucrose density centrifugation. A, A 254 profiles for sucrose density gradients. B, Agarose gel electrophoresis of RNA from each fraction with ethidium bromide staining to highlight 28 S, 18S and 5S ribosomal RNAs. Fractions 6–11 were pooled for polysomal RNA. Expression of mRNAs for Klf2 (C), Klf4 (D), Klf6 (E), Klf5 (F), Irs2 (H) or Il1rl1 (I) in total RNA and polysome RNA pools was analysed by qPCR. Results are expressed relative to levels in total RNA from unstimulated cells and are means ± S.E.M. for 3 or 4 independent preparations of myocytes. ⁎ p < 0.05, ⁎⁎ p < 0.001 relative to Control (total RNA); # p < 0.01 relative to ET-1 (total RNA) (one-way ANOVA with TUKEY post-test). G, Western blotting of Klf6 protein in cardiac myocytes exposed to ET-1 for the times indicated. A representative image is shown in the upper panel, with densitometric analysis in the lower panel (results are means ± S.E.M. for 3 independent myocyte preparations).

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Control, Centrifugation, Agarose Gel Electrophoresis, Staining, RNA Expression, Western Blot

Journal: Biochimica et Biophysica Acta

Article Title: Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

doi: 10.1016/j.bbamcr.2008.03.007

Figure Lengend Snippet: H 2 O 2 increases expression of Klf2, Klf4 and Klf6 mRNA in cardiac myocytes. A–C, Cardiac myocytes were exposed to 0.2 mM H 2 O 2 for the times indicated. RNA was extracted and expression of mRNAs for Klf2 (A), Klf4 (B), or Klf6 (C) analysed by qPCR. Results are expressed relative to unstimulated controls and are means ± S.E.M. for at least 4 independent preparations of myocytes. D and E, Cardiac myocytes were unstimulated (Control) or exposed to 0.2 mM H 2 O 2 (1 h) in the absence or presence of 5 μM SB203580 or 10 μM U0126 (D), or 50 μM LY294002 (E). Expression of mRNAs for Klf2 (pale grey bars), Klf4 (black bars), or Klf6 (dark grey bars) was analysed by qPCR. Results are expressed relative to levels in unstimulated myocytes and are means ± S.E.M. for 5 (D) or 7 (E) independent preparations of myocytes. ⁎ p < 0.001, # p < 0.01 relative to H 2 O 2 alone (one-way ANOVA with TUKEY post-test).

Article Snippet: Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution).

Techniques: Expressing, Control

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:

Article Title: pp32 Reduction Induces Differentiation of TSU-Pr1 Cells

doi:

Figure Lengend Snippet: Expression of IL-6 in TSU-Pr1 cells. TSU-Pr1 cells stably transfected with pp32 anti-sense express higher levels of IL-6 message as compared to parental TSU-Pr1 cells and vector-only control by RT-PCR analysis, which validates the cDNA microarray analysis (see Figure 6).

Article Snippet: Microarray Analysis of TSU-Pr1 Cell Lines This procedure was performed at The Johns Hopkins University Oncology Microarray facility by using a human glass 12K cDNA chip.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Microarray

Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) PTGS2 knockdown efficiency in NHEKs transfected with mock or PTGS2-specific siRNA. (B) qRT-PCR analysis of WNT7B mRNA expression in NHEKs (n=3) transfected with mock or PTGS2-specific siRNA, followed by treatment with or without poly(I:C), with the addition of prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), or 11β-prostaglandin F2α (11b-PGF2α). ‡Prostaglandin D2 induced significant cytotoxicity. (C) Representative western blot of Wnt7b protein expression in NHEKs treated as in Fig. 4B (n=3). (D) Western blot quantification for n=3 repeats performed as described in Fig. 4C. (E) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in mouse wounds injected with poly(I:C), with or without celecoxib. (F) Quantification of hair follicles in CSLM images from Fig. 4D (n=15–17). (G) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in mouse wounds injected with poly(I:C) and celecoxib, with or without dmPGE2. (H) Quantification of hair follicles in CSLM images from Fig. 4E (n=10–11). *denotes p<0.05.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Light Microscopy, Injection

Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in C57BL6/NJ mouse wounds injected with or without polyinosinic:polycytidylic acid (poly(I:C)). (B) Wnt ligands most strongly upregulated or downregulated in gene expression microarray comparing NHEKs treated with vehicle or poly(I:C), published under (GSE92646) (C) Representative immunofluorescent image of Wnt7b protein in C57BL6/NJ mouse wounds (scale bar = 100 μm). (D) qRT-PCR analysis of WNT7B mRNA expression in NHEKs (n=3) treated with various doses of poly(I:C). (E) Western blot analysis of Wnt7b protein expression in NHEKs treated with or without poly(I:C), with β-actin as a loading control.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Light Microscopy, Injection, Gene Expression, Microarray, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) qRT-PCR analysis of WNT7B mRNA expression in background (C57BL6/NJ) and TLR3 KO mouse wounds (n=8). (B) WNT7B mRNA expression in TLR3 KO mouse wounds injected with vehicle or recombinant IL-6 (n=8). (C) WNT7B mRNA expression in background or STAT3 KO mouse wounds (n=8). (D) WNT7B mRNA expression in NHEKs transfected with mock or TLR3-specific siRNA, followed by treatment with or without poly(I:C). *denotes p<0.05.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Quantitative RT-PCR, Expressing, Injection, Recombinant, Transfection

Journal: Stem cell research & therapy

Article Title: TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes.

doi: 10.1186/s13287-020-01711-w

Figure Lengend Snippet: Fig. 1 The TRF3 expression level is enhanced during ME differentiation of hESCs. a Microscope images for the morphology of ME differentiation. Scale bar = 200 μm. b The heat map of the expression pattern of early germ layer genes during the ME differentiation of hESCs based on the microarray analysis. The expression values in log2 scale were calculated and presented on the heat map with red representing highly abundant transcripts and green representing poorly abundant transcripts. n = 3 each. c qRT-PCR analysis of TBP, TRF2, and TRF3. Data are presented as mean ± SEM. n = 3 each. *p < 0.05, **p < 0.01 compared with the corresponding undifferentiated values

Article Snippet: Reintroduction of TRF3 into TRF3−/− hESCs The human TRF3 cDNA (RC211988, NM_199047, OriGene, USA) was cloned into pCDH-EF1-3×Flag-MCST2A-Puro, modified from pCDH-EF1-MCS-T2A-Puro (System Biosciences, CA, USA), and named as pCDHEF1-3×Flag-TRF3-T2A-Puro.

Techniques: Expressing, Microscopy, Microarray, Quantitative RT-PCR

Journal: Stem cell research & therapy

Article Title: TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes.

doi: 10.1186/s13287-020-01711-w

Figure Lengend Snippet: Fig. 3 The expression levels of pluripotent markers are comparable among the undifferentiated TRF3+/+, TRF3−/−-1, and TRF3−/−-2 hESCs. a qRT- PCR analysis of pluripotency markers OCT4 and NANOG. n = 3. b Flow cytometry analysis of OCT4 and SSEA4. c Immunocytochemical staining analysis of OCT4 and SSEA4. Similar results were obtained from three independent experiments. Scale bar = 50 μm. d Flow cytometry analysis of cell cycle in undifferentiated hESCs. n = 3. e Representative analysis of clones positive for alkaline phosphatase staining. Similar results were obtained from three independent experiments. Scale bar = 500 μm. Data are presented as mean ± SEM

Article Snippet: Reintroduction of TRF3 into TRF3−/− hESCs The human TRF3 cDNA (RC211988, NM_199047, OriGene, USA) was cloned into pCDH-EF1-3×Flag-MCST2A-Puro, modified from pCDH-EF1-MCS-T2A-Puro (System Biosciences, CA, USA), and named as pCDHEF1-3×Flag-TRF3-T2A-Puro.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Clone Assay

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: (A) GNAQQ209L transfected alone or combined with PKC δ or ε into 293FT cells increased Ras-GTP levels. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with a pan-Ras antibody. TPA stimulation was used a positive control. Expression levels of GNAQQ209L were monitored with Glu-Glu (EE) tag. PKC δ and ε were detected via HA tags.

Article Snippet: cDNA: HA-PKC α (catalytic domain) , addgene , 21234.

Techniques: Transfection, Western Blot, Positive Control, Expressing

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: cDNA: HA-PKC α (catalytic domain) , addgene , 21234.

Techniques: Recombinant, Activation Assay, Microarray, Fractionation, Expressing, Cell Culture

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: (A) GNAQQ209L transfected alone or combined with PKC δ or ε into 293FT cells increased Ras-GTP levels. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with a pan-Ras antibody. TPA stimulation was used a positive control. Expression levels of GNAQQ209L were monitored with Glu-Glu (EE) tag. PKC δ and ε were detected via HA tags.

Article Snippet: cDNA: HA-PKC δ(kinase dead) , addgene , 16389.

Techniques: Transfection, Western Blot, Positive Control, Expressing

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: cDNA: HA-PKC δ(kinase dead) , addgene , 16389.

Techniques: Recombinant, Activation Assay, Microarray, Fractionation, Expressing, Cell Culture