Journal: Animal Nutrition

Article Title: Lactobacillus johnsonii N5 from heat stress-resistant pigs improves gut mucosal immunity and barrier in dextran sodium sulfate-induced colitis

doi: 10.1016/j.aninu.2023.04.012

Figure Lengend Snippet: The phenotypic difference between the heat stress-susceptible and heat stress-resistant piglets in growth performance, inflammatory responses, and gut physiology. (A and B) The diarrhea indexes and body weight of Meishan piglets were recorded weekly during 8 weeks of cyclic heating treatment. The difference of body weight, diarrhea index was calculated as area under the curve. (C) The levels of TNF-α, IL-6, CXCL1 and IL-10 in porcine plasma. (D) Western blots showing the levels of HSP70, HSP27 and β-actin in the colonic tissues of HS-RES and HS-SUS pigs. (E) The expression of HSP70 and HSP27 normalized to β-actin expression. (F) qRT-PCR analysis of genes expression (fold change) of Hspa1a, Hspb1, HSF1 in the colonic tissues. HS-RES = heat stress resistant; HS-SUS = heat stress susceptible; TNF-α = tumor necrosis factor-alpha; IL-6 = interleukin-6; CXCL1 = chemokine (C–X–C motif) ligand 1; IL-10 = interleukin-10; HSP27 = heat shock proteins 27; HSP70 = heat shock proteins 70; Hspa1a = heat shock protein family A (HSP70) member 1A; Hspb1 = heat shock protein family B (small) member 1; HSF1 = heat shock factor 1. Data are presented as the means ± SEM, n = 6 piglets per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, using the two-tailed Student's t -test.

Article Snippet: For the detection, primary antibodies against HSP70 (bs-0244R, Bioss, Beijing, China), HSP27 (bs-0730R, Bioss, Beijing, China) and β-actin (sc-47778, Santa Cruz Biotechnology, New Mexico, USA), and the corresponding secondary antibodies (goat anti-rabbit IgG HRP, HX-2031, or goat anti-mouse IgG HRP, HX-2032; Huaxingbio, Beijing, China) were used.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 4. HN safeguards NP cells from erastin-induced ferroptosis via increasing HSP27 expression. (A-D and F-I) Western blot analysis of PTGS2, ACSL4, GPX4, MMP13, HSP27, and Col II and quantitative assessment in various experimental groups; (E and J) Immunofluorescence staining of MMP13, Col II, GPX4, HSP27, HN, and ACSL4 and quantitative analysis across different groups (scale bar = 50 μm); (K-L) ROS detecting in NP cells and quantitative analysis in different groups(scale bar = 50 μm); (M) Quantification of GSH levels in NP cells among different experimental groups; (N) Quantification of Fe2+ levels in NP cells among different experimental groups; (O) Quantification of MDA levels in NP cells among different experimental groups; (P-Q) Calcein/PI staining of NP cells in different experi mental groups (scale bar = 200 μm). *p < 0.05, **p < 0.01, ***p < 0.001 versus CTR or NC. The values are presented as the means ± SD from at least three in dependent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 6. HN suppresses ferroptosis in NP cells and activity of NF-κB and JAK2/STAT3 pathways depend on HSP27 expression partially. (A–B) Western blot of p-JAK2, JAK2, p-STAT3, STAT3, P65, and p-P65 and quantitative analysis when HSP27 was knocked down; (C–D) Immunofluorescence staining of p-JAK2, p-STAT3, and p-P65 when HSP27 was knocked down; (E-H and J-K)Western blot of HSP27, p-JAK2,JAK2, p-STAT3, STAT3, p-P65, P65, GPX4 PTGS2, MMP13, Col II, and ACSL4 in different groups; (I and L) Immunofluorescence staining of p-STAT3, p-P65, GPX4, MMP13, Col II, and ACSL4 in different groups (scale bar = 50 or 20 μm); (M−N) ROS detecting in NP cells and quantitative analysis in different groups(scale bar = 20 μm); (O) MDA level in NP cells in different groups; (P–Q) Calcein/PI staining of NP cells in different experimental groups (scale bar = 100 μm).*p < 0.05, **p < 0.01, ***p < 0.001 versus shNC. The values are presented as the means ± SD from at least three independent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: Activity Assay, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 8. HN alleviates IDD by inhibiting NP cells ferroptosis in rats. (A) The experimental procedure conducted in vivo; (B) ELISA for detecting FITC-HN In rat serum at the indicated time points; (C) Within 30 min after intranasal treatment, FITC-HN was detected associated with the IVD; (D–E) MRI images, H&E, and Safranin-O/fast green staining of rat discs in different groups (scale bar = 100 μm); (F) Histopathological score were utilized to assess the extent of IDD in different groups; (G–H) IHC staining of p-STAT3, p-P65, HSP27,and Rattin and quantitative analysis in different groups(scale bar = 100 μm); (I) Immunofluorescence of GPX4, ACSL4, Col II, and MMP13 in different groups (scale bar = 100 μm). *p < 0.05, **p < 0.01, ***p < 0.001 versus Sham or NS + IDD. Values are presented as the mean ± SD from at least three independent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Immunofluorescence

Journal: bioRxiv

Article Title: Allosteric MAPKAPK2 Inhibitors Improve Plaque Stability in Advanced Atherosclerosis

doi: 10.1101/2021.01.12.426264

Figure Lengend Snippet: ( A ) Primary mouse hepatocytes were treated with the indicated concentrations of TBX-1 for 1 h, followed by treatment with TBX-1 and forskolin for 4 h in serum-free media. RNA was assayed for G6pc mRNA by RT-qPCR (n = 3; mean ± SEM, p < 0.05). ( B ) Liver extracts from three different randomly selected subsets of TBX-1–treated Ldlr -/- mice were assayed for phopsho-hsp25 and β-actin by immunoblot. Densitometric quantification of the data are shown in the bar graphs (n = 5-6 mice/group; mean ± SEM, p < 0.05).

Article Snippet: Anti–phospho-hsp25 (CST, 2401) and anti-hsp25 (CST, 2442) antibodies were from Cell Signaling, and anti–β-actin antibody was from Abcam.

Techniques: Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Allosteric MAPKAPK2 Inhibitors Improve Plaque Stability in Advanced Atherosclerosis

doi: 10.1101/2021.01.12.426264

Figure Lengend Snippet: Mice were treated as in . ( A ) Aortic root sections from a randomly selected subset of Ldlr -/- mice treated with TBX-1 were co-immunostained for p-hsp25 and total hsp25. Phospho-hsp25 staining was quantified as MFI within hsp25 + cells. Data are presented relative to the average value obtained from the control group (n = 10 mice/group; mean ± SEM, p < 0.05). ( B-E ) Total lesion area and necrotic area were quantified from the aortic root sections of Ldlr -/- mice treated with TBX-1 ( B-C ) or TBX-2 ( D-E ) (n = 29 mice/group for TBX-1 and n = 28 mice/group for TBX-2; mean ± SEM, p < 0.05).

Article Snippet: Anti–phospho-hsp25 (CST, 2401) and anti-hsp25 (CST, 2442) antibodies were from Cell Signaling, and anti–β-actin antibody was from Abcam.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Aging associated altered response to intracellular bacterial infections and its implication on the host

doi: 10.1101/2020.09.15.298158

Figure Lengend Snippet: A . Bacterial CFU at 16h post invasion in vehicle, NAC and AMG treated senescent HeLa cells. B . p38 MAPK inhibition decreases bacterial proliferation. Senescent HeLa cells were infected in the presence of a specific p38MAPK inhibitor, SB 202190 (SB) and bacterial CFU was determined at 16 h post invasion. C . Western blot of p38 activity by monitoring phosphorylation status of Hsp27, a downstream substrate of p38MAPK in infected and uninfected senescent cells, treated with vehicle or SB202190. D . Analysis of changes in NO levels in SB202190 treated senescent cells by Griess assay. E . Expression analysis of NOS2 in senescent cells treated with SB202190 by qRT-PCR. Values are normalized to β-actin and then wrt vehicle treated cells to obtain fold changes. F . Effect of co-inhibition of iNOS by AMG and p38 by SB202190 on intracellular bacterial proliferation. The data represents mean ± SEM from atleast three independent experiments. Statistical significance of differences was analysed by Mann-Whitney U test, * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: All the primary antibodies were from CST (Cell Signalling Technology Inc., USA) and used at 1:1000 dilution overnight at 4°C for probing protein levels viz. phospho-Hsp27 (Cat No. 9709), Hsp27 (Cat No. 95357) GAPDH (Cat No. 2118) and phospho-Chk2 (Cat no. 2197).

Techniques: Inhibition, Infection, Western Blot, Activity Assay, Griess Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY

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: International Journal of Oncology

Article Title: Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine

doi: 10.3892/ijo.31.6.1345

Figure Lengend Snippet: Figure 3. Comparison of spots of HSP27 between KLM1 and KLM1-R. The 2-DE pattern of KLM1 is on the left and that of KLM1-R is on the right. The spot numbers correspond to those in Fig. 2. HSP27 was shown to be up- regulated in KLM1-R cells compared with KLM1 cells.

Article Snippet: At 24 h after seeding, either specific HSP27 siRNA (Santa Cruz Biotechnology) or control siRNA (Santa Cruz Biotechnology) was added at a final concentration of 520 nM and incubation was performed for 30 h. Then the medium was exchanged for 2.0 ml of fresh growth medium containing 10% FCS and cells were incubated for 24 h. For the MTT assay, cells were trypsinized and transferred to 96-well plates.

Techniques: Comparison

Journal: International Journal of Oncology

Article Title: Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine

doi: 10.3892/ijo.31.6.1345

Figure Lengend Snippet: Figure 4. Immunoblotting of HSP27. HSP27 was shown to be up-regulated in KLM1-R cells compared with KLM1 cells. When gemcitabine-resistant KLM1-R cells were treated with specific siRNA targeting HSP27, a significant reduction of HSP27 protein expression was detected compared with negative control siRNA-treated KLM1-R cells and normal KLM1-R cells.

Article Snippet: At 24 h after seeding, either specific HSP27 siRNA (Santa Cruz Biotechnology) or control siRNA (Santa Cruz Biotechnology) was added at a final concentration of 520 nM and incubation was performed for 30 h. Then the medium was exchanged for 2.0 ml of fresh growth medium containing 10% FCS and cells were incubated for 24 h. For the MTT assay, cells were trypsinized and transferred to 96-well plates.

Techniques: Western Blot, Expressing, Negative Control

Journal: International Journal of Oncology

Article Title: Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine

doi: 10.3892/ijo.31.6.1345

Figure Lengend Snippet: Figure 5. Sensitivity of HSP27-silenced KLM1-R cells to gemcitabine. The HSP27-silenced KLM1-R cells showed increased drug sensitivity as well as KLM1.

Article Snippet: At 24 h after seeding, either specific HSP27 siRNA (Santa Cruz Biotechnology) or control siRNA (Santa Cruz Biotechnology) was added at a final concentration of 520 nM and incubation was performed for 30 h. Then the medium was exchanged for 2.0 ml of fresh growth medium containing 10% FCS and cells were incubated for 24 h. For the MTT assay, cells were trypsinized and transferred to 96-well plates.

Techniques:

Journal: International Journal of Oncology

Article Title: Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine

doi: 10.3892/ijo.31.6.1345

Figure Lengend Snippet: Figure 6. Immunohistochemistry of HSP27 in pancreatic cancer tissues and its correlation with survival rate of patients. (a) Tissue from a patient with progressive disease (PD). Fifty-one percent of the cancerous area was immunopositive (x400). (b) Tissue from a patient with stable disease (SD). Eleven percent of the cancerous area was immunopositive (x400). (c) The positive rate of HSP27 in PD and SD. The PD group's ratio of positive for HSP27 was higher than that of the SD group. (d) Immunohistochemistry positive rate of HSP27 and patient survival. Patients whose positive rate was >30% had a shorter survival than those with a rate <30%.

Article Snippet: At 24 h after seeding, either specific HSP27 siRNA (Santa Cruz Biotechnology) or control siRNA (Santa Cruz Biotechnology) was added at a final concentration of 520 nM and incubation was performed for 30 h. Then the medium was exchanged for 2.0 ml of fresh growth medium containing 10% FCS and cells were incubated for 24 h. For the MTT assay, cells were trypsinized and transferred to 96-well plates.

Techniques: Immunohistochemistry

Journal: Experimental and therapeutic medicine

Article Title: Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model.

doi: 10.3892/etm.2022.11274

Figure Lengend Snippet: Figure 4. ALR‑S decreases ROS generation during platelet activation. (A) Platelet intracellular ROS level was determined using flow cytometry. Platelets loaded with H2DCFDA (50 µmol/l) were incubated with ALR‑S or vehicle for 10 min and then stimulated with CRP (2 µg/ml), thrombin (0.025 U/ml), or ADP (10 µM) for 5 min. *P<0.05 and **P<0.01 vs. control. (B) Western blotting was performed to analyze the effects of ALR‑S on phosphorylated ERK1/2, p38 and HSP27 during platelet activation in an aggregometer. (C) Representative aggregation traces of washed platelets in response to collagen, thrombin or ADP. Washed platelets were preincubated with ALR‑S (0, 20, 60 and 200 µg/ml), respectively. H2O2 (20 µM) was added before aggregation was started, and traces were recorded by a Chrono‑log aggregometer under stirring. ALR‑S, saccharides from Arctium lappa L. root; p38, p38 mitogen‑activated protein kinase; HSP27, heat shock protein 27; Col, collagen; Thr, thrombin; MFI, mean fluorescence intensity; ROS, reactive oxygen species; CRP, collagen‑related peptide.

Article Snippet: Antibodies against phospho‐ERK1/2 (catalog no. 4370S), phospho‐p38 (catalog no. 4511S), phospho‐HSP27 (catalog no. 2401S), total‐p38 (catalog no. 8690S), total‐ERK (catalog no. 4695S) and HSP27 (catalog no. 95357S) were purchased from Cell Signaling Technology Inc. β‐actin monoclonal antibody (catalog no. 66009‐1‐Ig) was obtained from ProteinTech Group, Inc.

Techniques: Activation Assay, Flow Cytometry, Incubation, Control, Western Blot, Fluorescence