Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: Microarray analysis of the hippocampi of 6-month-old WT and hTau MaptKO ( Duke ) mice. (A) Total RNA from hippocampi of 6-month-old WT and hTau MaptKO ( Duke ) mice was hybridized to MouseWG-6 v2.0 Expression BeadChip (Illumina ® ). Expression of 64 genes that were significantly altered in hTau MaptKO ( Duke ) mice compared to WT is shown. Values displayed as fold change in expression level: up-regulated (red) and down-regulated (blue) genes. Data represents mean fold change from three mice per genotype. (B) Gene interaction network analysis (using Metacore analytical suite) for the 64 significantly altered genes in hTau MaptKO ( Duke ) mice compared to WT mice. The genes with red (or blue) next to their graphic key is either up- (or down-) regulated. As expected, endogenous mouse tau (MAPT) is one of the most down-regulated genes. Other genes relevant to neuronal function or neurological disease include Prkca, Mecp2, Strn4, Slc40a1, Pold2, Pcsk2 (up-regulated) and Krt12, Lass1, Plat and Nrxn1 (down-regulated). Many of the altered genes are regulated by the transcription factor SP1. (C) Venn diagrams showing GO’s biological processes when all 64 genes are categorized for top biological processes segregate into three distinct Venn diagrams: Group 1: anatomical structure development – 17 altered genes out of total 47, i.e., 17/47; cellular nitrogen compound metabolic process – 15/47; biosynthetic process 13/47. Group 2: cell death – 9/47; signal transduction 8/47; response to stress 8/47. Group 3: neurological system 5/47, cell cycle 2/47 and immune system processes 4/47. Note the genes written in red (or blue) are significantly up- (or down-) regulated.

Article Snippet: For the gene expression analysis: Total RNA (50 ng/μL) was converted to cDNA using the High Capacity cDNA Reverse Transcription kit and amplified using specific TaqMan probes ( gene expression marker: Mecp2 Mm01193535_m1; MAPT Hs00902194_m1) GAPDH was used as a house-keeping gene for normalization.

Techniques: Microarray, Expressing, Transduction

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 expression and phosphorylation is up-regulated in 6-month-old hTau MaptKO ( Duke ) mice. (A) qRT-PCR analysis showing statistically significant ( ∗ p < 0.05; unpaired t- test; n = 4 WT, and n = 4 for hTau MaptKO ( Duke ) mice; mean + SEM) up-regulation of MECP2 in the hemi-brains of 6-month-old hTau MaptKO ( Duke ) vs. WT mice. (B) Note the regional differences in the expression of MECP2 in the hippocampus (HIP), cortex (CX), and rest of the brain (ROB) that are devoid of CX and HP. (C) Another gene ( Vat1l ) that was increased in our whole genome microarray analysis also showed modest up-regulation in its mRNA levels in the brains of hTau MaptKO ( Duke ) mice compared to age-matched WT mice. (D,E) Western blot analysis showing a trend toward increased levels for phospho(p)-Ser80 MECP2/total (t) MECP2 and tMECP2/GAPDH [ p = 0.08; unpaired t- test; n = 3, all females for WT and n = 10, three females and six males for hTau MaptKO ( Duke ) ] in 6-month-old hTau MaptKO ( Duke ) versus WT mice; mean + SEM). (F) Double IF and confocal microscopy analysis revealing a modest increase in pMECP2 in the CA3 region of HP in 6-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. Scale bar 25 μm.

Article Snippet: For the gene expression analysis: Total RNA (50 ng/μL) was converted to cDNA using the High Capacity cDNA Reverse Transcription kit and amplified using specific TaqMan probes ( gene expression marker: Mecp2 Mm01193535_m1; MAPT Hs00902194_m1) GAPDH was used as a house-keeping gene for normalization.

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Microarray, Western Blot, Confocal Microscopy

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 phosphorylation is up-regulated in 12-month-old hTau MaptKO ( Duke ) mice and in human AD brain. (A,B) Western blot analysis showing pMECP2/GAPDH ratio significantly higher [ ∗ p < 0.05; unpaired t- test; n = 3 for hTau MaptKO ( Duke ) versus WT mice; all three males for WT; two males and one female for hTau MaptKO ( Duke ) ; mean + SEM] in the hippocampus of 12-month-old hTau MaptKO ( Duke ) mice compared to WT controls. No alteration in the tMECP2/GAPDH ratio in the hippocampus of 12-month-old hTau MaptKO ( Duke ) mice compared to controls. (C) Significantly elevated pMECP2 immunoreactive specks within the nucleus of CA3 hippocampal neurons of 12-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. (D) Double IF and confocal microscopy analysis shows a modest increase in the pMECP2 in the CA3 neuronal layer of 12-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. (E–G) Confocal projections (in E,G ) and bright field images show elevated pMECP2 immunoreactivity and co-localization with nuclear stain DAPI (in E,G ) or peri-vascular labeling (in F ) specifically in the Layer III of temporal cortex of human AD brain autopsy sections compared to the to non-AD healthy control subject. Orthogonal view in (G) shows pMECP2 labeling to be nuclear or peri-nuclear in the human AD cortex. Scale bar 10 μm (in C,F ); 25 μm (in D,G ); 100 μm (in E ).

Article Snippet: For the gene expression analysis: Total RNA (50 ng/μL) was converted to cDNA using the High Capacity cDNA Reverse Transcription kit and amplified using specific TaqMan probes ( gene expression marker: Mecp2 Mm01193535_m1; MAPT Hs00902194_m1) GAPDH was used as a house-keeping gene for normalization.

Techniques: Phospho-proteomics, Western Blot, Confocal Microscopy, Staining, Labeling, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 regulates tau pathology in vitro . N2a cells transiently transfected with human tau 0N3R isoform (‘+Tau’) or a control plasmid (‘-Tau’) were nucleofected with siRNA [scramble siRNA (siScr) or MECP2 siRNA]. After 24 h of siRNA nucleofection, the cells were harvested and probed for AT180, PHF-1, Tau5, Tau12, and T-MECP2. (A,B) Note that siMECP2 significantly reduced levels of MECP2 in both ‘-Tau’ and ‘+Tau’ N2a cells ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM). (C,D) siMECP2 treatment also significantly ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM) reduced the levels of both total tau (Tau5/GAPDH) and human tau (Tau12/GAPDH) ratios in the ‘+Tau’ N2a cells compared to scramble siRNA treated conditions. (E–H) siMECP2 knockdown resulted in statistically significant ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM) increase and decrease in AT180/Tau5 and PHF1/Tau5 ratios, respectively. Note that the ratio for β-actin/GAPDH was not altered either in ‘-Tau’/‘+Tau’ conditions or with/without siMECP2 conditions.

Article Snippet: For the gene expression analysis: Total RNA (50 ng/μL) was converted to cDNA using the High Capacity cDNA Reverse Transcription kit and amplified using specific TaqMan probes ( gene expression marker: Mecp2 Mm01193535_m1; MAPT Hs00902194_m1) GAPDH was used as a house-keeping gene for normalization.

Techniques: In Vitro, Transfection, Control, Plasmid Preparation, Knockdown

Journal: Bioactive Materials

Article Title: Bioinspired engineering ADSC nanovesicles thermosensitive hydrogel enhance autophagy of dermal papilla cells for androgenetic alopecia treatment

doi: 10.1016/j.bioactmat.2024.02.023

Figure Lengend Snippet: JAM-A protein plays a core role in ADSC-NVs-mediated AGA hair regrowth. A) Volcano plot of the GSE184501 microarray data set, and JAM-A significantly downgraded. B) Change in JAM-A expression. C) Relative expression of JAM-A. D) Viability of DPC cell lines treated with DHT for 48 h. E) Statistical data on the proportion of senescent cells in each group was counted in the same random area. F) Effect of DHT on DPC cell line migration at every indicating time; scale bar: 50 μm. G) Relative migration of DHT-injured DPC cell lines in each group. H) Apoptosis cells of DPC cell lines were injured with macrophages for 48 h. I) Statistical data on the proportion of apoptosis cells in each group. J) Change in LC3, AKT, AKT phosphorylation, and p62 expression. K) Relative expression of LC-3II, p62, and AKT phosphorylation. L) Change in Cyclin D1 and β-catenin expression. M) Relative expression of Cyclin D1 and β-catenin. (n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Subsequently, they were incubated with CD63 (1:1000, ab134045, Abcam) or TSG101 (1:1000, ab125011, Abcam) or Calnexin (1:1000, ab133615, Abcam) or β-catenin (1:1000, ab32572, Abcam) or LC3B (1:1000, ab192890, Abcam) or GAPDH (1:4000, 60,004-1-lg, Proteintech) or α-tubulin (1:1000, 2144S, Cell Signaling Technology) or JAM-A (1:1000, 36–1700, Invitrogen) or p62 (1:1000, ab109012, Abcam) or β-actin (1:1000, GB11001-100, Servicebio) or AKT (1:1000, 4685, Cell Signaling Technology) or phospho-Akt (1:1000, 4060S, Cell Signaling Technology) or Cyclin D1 (1:1000, 60186-1-Ig, Proteintech) primary antibodies overnight at 4 °C.

Techniques: Microarray, Expressing, Migration, Phospho-proteomics

Journal: Bioactive Materials

Article Title: Bioinspired engineering ADSC nanovesicles thermosensitive hydrogel enhance autophagy of dermal papilla cells for androgenetic alopecia treatment

doi: 10.1016/j.bioactmat.2024.02.023

Figure Lengend Snippet: Protective effect of JAM-A OE @NV on JAM-A-downregulated DPC cell line injured by DHT and macrophages. A) Change in JAM-A expression. B) Relative expression of JAM-A. C) Viability of the DHT-injured, downregulated DPC cell line treatment with JAM-A@NV for 48 h. D) Statistical data on the proportion of senescent cells in each group was counted in the same random area. E) Effect of JAM-A@NV on the DHT-injured, downregulated DPC cell line migration at 24 h; scale bar: 50 μm. F) Relative migration of the DHT-injured, downregulated DPC cell line in each group. G) Apoptosis cells of the macrophages injured by downregulated DPC cell line treatment with JAM-A@NV for 48 h. H) Statistical data on the proportion of apoptosis cells in each group. I) Change in LC3, AKT, AKT phosphorylation, and p62 expression. J) Relative expression of LC-3II, p62, and AKT phosphorylation. K) Change in Cyclin D1 and β-catenin expression. L) Relative expression of Cyclin D1 and β-catenin. (n = 6 per group, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Subsequently, they were incubated with CD63 (1:1000, ab134045, Abcam) or TSG101 (1:1000, ab125011, Abcam) or Calnexin (1:1000, ab133615, Abcam) or β-catenin (1:1000, ab32572, Abcam) or LC3B (1:1000, ab192890, Abcam) or GAPDH (1:4000, 60,004-1-lg, Proteintech) or α-tubulin (1:1000, 2144S, Cell Signaling Technology) or JAM-A (1:1000, 36–1700, Invitrogen) or p62 (1:1000, ab109012, Abcam) or β-actin (1:1000, GB11001-100, Servicebio) or AKT (1:1000, 4685, Cell Signaling Technology) or phospho-Akt (1:1000, 4060S, Cell Signaling Technology) or Cyclin D1 (1:1000, 60186-1-Ig, Proteintech) primary antibodies overnight at 4 °C.

Techniques: Expressing, Migration, Phospho-proteomics

Journal: Molecular Cancer Therapeutics

Article Title: Suppression of Extracellular Vesicle VEGF-C–mediated Lymphangiogenesis and Pancreatic Cancer Early Dissemination By a Selective HDAC1/2 Inhibitor

doi: 10.1158/1535-7163.mct-20-0963

Figure Lengend Snippet: Figure 2. Exogenous expression of DUSP2 inhibits pancreatic tumor formation. A, Comparative transcriptome profiling of HDAC inhibitors. Microarray analysis of upregulated gene expression in TSA (T) and sodium butyrate (B) treated cells. Two time-points (12 and 48 hours) were included for analysis. B, RT-qPCR of DUSP2 expression in MIA PaCa-2 cells treated with SAHA (S, 4 mmol/L) and B390 (B, 0.25 and 1 mmol/L) for 24 hours. CYCLOPHILIN B (PPIB) was used as internal control. C, Western blotting result for the expression of DUSP2, ERK1/2 phosphorylation (pERK) and expression (tERK) in MIA PaCa-2 cells treated with control or B390 (0.25 and 1 mmol/L) for 24 hours. GAPDH were detected as internal control for whole cell lysate. D, Representative images show the fluorescent signal of GFP in GFP or DUSP2- GFP–transfected MIA PaCa-2 cells (left). Western blotting for the expression of DUSP2 and ERK1/2 in MIA PaCa-2 cells transfected with GFP or DUSP2-GFP expression vector. GAPDH is the internal control for whole-cell lysate (right). E, Representative images show crystal violet-stained cells growing on 24-well plates for 48 hours. Equal numbers of MIA PaCa-2 cells were plated on 24-well plates and transfected with GFP or DUSP2-GFP expression vectors. This experiment was repeated by MTT assay with similar result (right). F, Western blotting for the expression of cleaved caspase 3 (cCas3) in MIA PaCa-2 cells. MIA PaCa-2 cells were transfected with GFP or DUSP2-GFP and treated with DMSO (D) or B390 (1 mmol/L) for 24 hours. b-Actin is the loading control. G, Growth curve (left), tumor mass, and representative image (right) show forced expression of DUSP2 abolished tumor growth in mice pancreas. GFP or DUSP2-GFP MIA PaCa-2 cells were labeled with luciferase and injected into mouse pancreas (n ¼ 6 mice per group). Tumor masswas followed by IVIS imaging. 42 days after inoculation, mice were sacrificed, tumors and pancreases were collected. H, IHC of Ki67 and cleaved caspase 3 in control (shCtrl) and DUSP2 KD (shDUSP2) PANC-1 tumors. , P < 0.05; , P < 0.001.

Article Snippet: Antibodies against DUSP2 (Santa Cruz Biotechnology, catalog no. sc-32776, RRID:AB_2094883), HDAC1 (Cell Signaling Technology, catalog no. 5356, RRID: AB_10612242), HDAC2 (Cell Signaling Technology, catalog no. 5113, RRID:AB_10624871), phospho-p44/42 MAPK (Cell Signaling Technology, catalog no. 4370, RRID:AB_2315112), p44/42MAPK (Cell Signaling Technology, catalog no. 4696, RRID:AB_390780), VEGF-C (GeneTex, GTX113574, RRID:AB_10620764), CD9 (ProteinTech, 60232–1, RRID: AB_11232215),HSP70 (SantaCruzBiotechnology, catalogno. sc-32239), GAPDH (GeneTex, GTX100118, RRID:AB_1080976) were used.

Techniques: Expressing, Microarray, Gene Expression, Quantitative RT-PCR, Control, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Staining, MTT Assay, Labeling, Luciferase, Injection, Imaging

Journal: Molecular Cancer Therapeutics

Article Title: Suppression of Extracellular Vesicle VEGF-C–mediated Lymphangiogenesis and Pancreatic Cancer Early Dissemination By a Selective HDAC1/2 Inhibitor

doi: 10.1158/1535-7163.mct-20-0963

Figure Lengend Snippet: Figure 3. Loss of Dusp2 in the pancreas facilitates the progression of Kras-driven PanIN progression. A, Schematic diagram of breeding KC and KDC transgenic mouse model. B, Histology of pancreas from 2-month old KDC mice and WT littermate. C, H&E staining of pancreas in 6-month old WT (a and b), KC mice (c and d), and KDC mice (e and f). Normal pancreatic ducts (b) show single layer of epithelial cells. Pancreas of KC shows ADM and PanINs while pancreas of KDC display larger area of abnormalities, including ADM, PanINs, and carcinoma. Early PanIN display papillary or micropapillary projections. Advanced PanIN has epithelial cells budding into the lumen and necrosis in the lumen. Carcinoma displays nuclear abnormalities and glands embedded in tumor stroma. D, IHC staining of phosphor- p44/42 MAPK (pERK) in 6-month old pancreas from control, KC, and KDC mice.

Article Snippet: Antibodies against DUSP2 (Santa Cruz Biotechnology, catalog no. sc-32776, RRID:AB_2094883), HDAC1 (Cell Signaling Technology, catalog no. 5356, RRID: AB_10612242), HDAC2 (Cell Signaling Technology, catalog no. 5113, RRID:AB_10624871), phospho-p44/42 MAPK (Cell Signaling Technology, catalog no. 4370, RRID:AB_2315112), p44/42MAPK (Cell Signaling Technology, catalog no. 4696, RRID:AB_390780), VEGF-C (GeneTex, GTX113574, RRID:AB_10620764), CD9 (ProteinTech, 60232–1, RRID: AB_11232215),HSP70 (SantaCruzBiotechnology, catalogno. sc-32239), GAPDH (GeneTex, GTX100118, RRID:AB_1080976) were used.

Techniques: Transgenic Assay, Staining, Immunohistochemistry, 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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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: Following siRNA transfection and P. gingivalis infection, GECs were fixed with 10% NBF, permeabilized by 0.1% Triton X-100, and stained for 1 h at RT with anti- P. gingivalis ATCC 33277 mouse or rabbit antibody (1:1000), anti-HSp27 mouse (Santa Cruz Biotechnology, sc-13132, 1:250) or goat (R&D Systems, AF15801, 1:250) antibody, anti-Beclin 1 rabbit (Cell Signaling Technology, 3495; 1:500 or sheep (R&D Systems, AF5295; 1:250) antibody, ATG14 mouse antibody (Cell Signaling Technology, 96752; 1:250) or anti-LC3C rabbit antibody (ProteinTech, 18726-1-AP: 1:500).

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

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Heterozygosis of Usp16 in mammary tissue promotes Wnt-driven in vitro and in vivo cell expansion. ( a ) FACS analysis shows a higher basal to luminal cell ratio in the preneoplastic mammary gland of virgin MMTV-Wnt1-Usp16 +/− mice. On the left, representative FACS plots of Lin − (Ter119 − CD45 − CD31 − ) mammary cells for the indicated genotypes. On the right, quantification of basal/luminal cell ratio. Each dot represents an individual mouse. ( b – c ) Histological analyses of preneoplastic mammary glands reveal an increase in the number and area of ducts derived from MMTV-Wnt1-Usp16 +/− mice. The graph shows the average of six slides analyzed per animal, two animals per group. Quantification was performed with ImageJ software. On panel C, two representative pictures per genotype are shown. Keratin 8 and Keratin 14 were used to mark luminal and basal cell layers, respectively. The white bar scale is 100 μm. ( d ) FACS-sorted epithelial cells from MMTV-Wnt1-Usp16 +/− preneoplastic mammary glands form more colonies in vitro compared to control mice (n = 3 per group). Shown is passage P1. ( e ) Usp16 +/− sorted mammary epithelial cells show an increased induction of Axin2 mRNA levels 16 hours after Wnt3A stimulation (50 ng/ml). Three independent experiments were performed. (f) The mammary epithelial TCF-GFP + frequency is increased in Usp16 +/− compared to wt TCF-GFP animals after one passage in vitro . Each dot represents an individual animal. Unpaired T-test shows a two-tailed pvalue < 0.01. ( g ) Limiting dilution analysis (ELDA) shows an increased frequency of mammary repopulating cells in Usp16 +/− compared to wt breast tissue. Four rounds of transplants were performed. The table shows the stem cell frequency and the 95% confidence interval between lower and upper values. ( h ) Usp16 +/− cells were able to engraft better than wt cells in secondary recipients. Two independent experiments were performed. Each circle represents a recipient. In black, the estimated area occupied by the mammary tree in the fat pad two months after transplantation.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques: In Vitro, In Vivo, Derivative Assay, Software, Control, Two Tailed Test, Transplantation Assay

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Cdkn2a mediates Usp16 response to Wnt3a. ( a ) Basal cell expansion is observed in the mammary gland of Cdkn2a +/− mice. A Graph quantifying the basal/luminal ratio is shown. Each dot represents an individual mouse. ( b ) Cdkn2a +/− sorted basal cells show an increased induction of Axin2 mRNA levels after 16 hours of Wnt3A stimulation (50 ng/ml). Two independent experiments were performed for a total of 4 animals per group. ( c ) Terminal Tip Fibroblasts (TTFs) derived from Cdkn2a +/− animals show an increase response to two different doses of Wnt3a (20 ng/ml and 50 ng/ml). The graph shows activation of a Top/Flash reporter for Wnt activity normalized by Renilla expression, co-transfected along with the reporter. ( d ) TTFs derived from Cdkn2a −/− animals and transfected with a vector expressing a Usp16 transgene or a control vector were analyzed for their ability to activate the Top-flash reporter. No differences were observed with two different doses of Wnt3a (20 ng/ml and 100 ng/ml). ( e ) MEFs cells were transiently transfected with two individual siRNAs targeting Bmi1. Activation of 6KD Wnt reporter is shown, normalized by Renilla expression. Cells were treated with 100 ng/ml of Wnt3a for 16 hours. ( f ) Two different human foreskin cultures were transiently transfected with siRNA targeting USP16, p16 Ink4a or a scramble sequence. USP16 and p16 Ink4a downregulation promotes the induction of AXIN2 mRNA expression 24 hours after treatment with 100 ng/ml of Wnt3a.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques: Derivative Assay, Activation Assay, Activity Assay, Expressing, Transfection, Plasmid Preparation, Control, Sequencing

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Usp16 and p16 Ink4a modulates Rspo-mediated LRP6 phosphorylation. ( a ) MEFs cells were transiently transfected with siRNAs targeting Usp16 or p16 Ink4a and treated with or without Wnt. Representative staining for βcatenin (in red) and DAPI (in blue) is shown on the left. On the right, the graph shows quantification of βcatenin intensity. Treatment with siRNA against Usp16 or p16 Ink4a increases levels of βcatenin upon treatment with 50 ng/ml of Wnt3a. Experiments were repeated twice with two different MEF cultures. ( b ) Usp16 +/− mammary epithelial cells, both untreated and treated with Wnt3a, express higher levels of Rspo1 and Rspo2 (n = 3 per group). ( c ) FACS-sorted mammary basal cells heterozygous for Usp16 express higher levels of Lgr5 upon treatment with Wnt3a (n = 4 per group). ( d ) Extracts derived from MEF cells and treated with the indicated siRNA were tested by western blot. Phospho-LRP6 immunoblots are shown on the left panel. βactin is used as loading control. A white line has been introduced between samples for clarity, but the image has not been cropped. On the right, the graph shows quantification of phospho-LRP6 intensity. Experiments were repeated four times.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques: Phospho-proteomics, Transfection, Staining, Derivative Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: Usp16 trisomy affects the Wnt pathway in Down’s Syndrome. ( a ) SYBR Green analyses revealed a marked decrease in the expression of a Top-Gal reporter of Wnt activity in mammary epithelial cells FACS-sorted from Ts65Dn mice compared to wt animals (n = 3 per group). ( b ) TTFs derived from animals with the indicated genotypes were analyzed for their ability to activate the Top-flash reporter in response to 20 ng/ml of Wnt3a. Data were normalized based on induction in wt animals. Wt and Ts65Dn/Usp16 +/− cells activate Top-Flash more efficiently than Ts65Dn-derived cells. (wt, n = 5; Ts65Dn, n = 5; Ts65Dn/Usp16 +/− , n = 2). ( c ) Microarray analysis shows clustering of wt, Ts65Dn and Ts65Dn/Usp16 +/− RNA expression based on a Wnt signature of genes differentially expressed between wt and Ts65Dn cells (p < 0.05). (n = 2 per group). Ts65Dn/Usp16 +/− RNA expression (samples in the middle) clusters with wt expression (samples on the right). ( d ) FACS-sorted epithelial cells from Ts65Dn mammary glands form less colonies in vitro compared to their wt counterpart. Axin2 heterozygosis increases the ability of Ts65Dn cells to form colonies (n = 3 animals per group; n = 2 for wt animals). Shown is passage P0. ( e , f ) Real time PCR analyses showed that human DS foreskin fibroblasts are less responsive to Wnt3a than cells derived from healthy patients in the activation of AXIN2, RSPO2 and RSPO3 (healthy, n = 4: DS, n = 3).

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques: SYBR Green Assay, Expressing, Activity Assay, Derivative Assay, Microarray, RNA Expression, In Vitro, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: General mechanism of Wnt induction. ( a ) Proposed mechanism of interaction between Usp16 and the Wnt pathway. Bmi-1 and Usp16 control the level of ubiquitination of histone H2A. The balance between these two antagonistic proteins determines the levels of activation of Cdkn2a . Cdkn2a acts as a negative regulator of the Wnt pathway. Other mechanisms linking Bmi1 and Usp16 to Wnt activation are also possible. ( b ) During aging, increased tissue senescence and increased expression levels of p16 INK4a have been observed. At the same time, there is a concurrent decline in stem cell self renewal and Wnt activation. These events are correlated to a change in balance between Bmi1 - promoting self-renewal and Wnt activation - and Usp16 - promoting cellular senescence and Cdkn2a expression.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques: Control, Ubiquitin Proteomics, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: List of Taqman assays (mouse and human-specific) used for this study. Probes were purchased from Applied Biosystem.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques:

Journal: Scientific Reports

Article Title: Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

doi: 10.1038/s41598-018-34562-w

Figure Lengend Snippet: List of siRNA (mouse and human-specific) used for this study. siRNA were purchased from Dharmacon.

Article Snippet: Usp16 , Hs01062190_m1 , Mm_00470393.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Cardioprotective Effects of Salvianolic Acid A on Myocardial Ischemia-Reperfusion Injury In Vivo and In Vitro

doi: 10.1155/2012/508938

Figure Lengend Snippet: Protein factors whose phosphorylation status altered in H 2 O 2 -induced H9c2 cells. H9c2 cells were pretreated with or without SAA (16 nM) for 24 h prior to incubation of cells with H 2 O 2 for 1 h. Thereafter, proteins were extracted and analyzed. The phosphor-antibody microarray identified a list of protein factors whose phosphorylation status altered in H 2 O 2 -induced H9c2 cells treated with or without SAA. The signal intensities of phosphorylated proteins and the total proteins were determined. The ratio of each protein was determined as the ratio between the percentages of phosphorylated proteins of two groups. The phosphorylation induction ratio of the control group was given a value of 1.

Article Snippet: Phosphospecific protein microarray (YT-PAP247) was a product of Full Moon BioSystems (USA).

Techniques: Phospho-proteomics, Incubation, Microarray, Control

Journal:

Article Title: Microarray and Real-Time PCR Analysis of Adrenal Gland Gene Expression in the 7-day-old Rat: Effects of Hypoxia from Birth

doi: 10.1152/physiolgenomics.00245.2006

Figure Lengend Snippet: TaqMan Gene Expression Assays Used for Real-Time PCR

Article Snippet: Differences in gene expression, expressed as fold-change, were calculated using the 2 −ΔΔCt method using 18S as the internal control. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Symbol Assay ID# Cytochrome P450, subfamily 1B, polypeptide 1 Cyp1b1 Rn00564055_m1 Guanosine monophosphate reductase Gmpr Rn00589361_m1 Xanthine dehydrogenase Xdh Rn00567654_m1 Cadherin 1 Cdh1 Rn00580109_m1 Phospholipase A2, group IVA (cytosolic, Ca-dependent) Pla2g4a Rn00591916_m1 Aldehyde oxidase Aox1 Rn00571242_m1 Regulator of G-protein signaling protein 2 Rgs2 Rn00584932_m1 Carnitine palmitoyltransferase 2 Cpt2 Rn00563995_m1 Steroid 5 alpha-reductase 1 Srd5a1 Rn00567064_m1 Enolase 1, alpha Eno1 Rn00820594_g1 N-ethylmaleimide sensitive factor Nsf Rn00572694_m1 Cytochrome P450, subfamily 11B, polypeptide 3 Cyp11b3 Rn00822066_g1 Steroidogenic acute regulatory protein StAR Rn00580695_m1 Cytochrome P450, subfamily 11A Cyp11a Rn00568733_m1 Cytochrome P450, subfamily 21A, polypeptide 1 Cyp21a1 Rn00588996_g1 3-hydroxy-3-methylglutaryl-Coenzyme A reductase Hmgcr Rn00565598_m1 Low density lipoprotein receptor Ldlr Rn00598438_m1 Vascular endothelial growth factor Vegf Rn00582935_m1 Hypoxia-inducible factor 1 alpha Hif1a Rn00577560_m1 Open in a separate window Each Gene Expression Assay consisted of a mix of sequence specific forward/reverse primers and a FAM-labeled probe.

Techniques: Gene Expression

Journal:

Article Title: Microarray and Real-Time PCR Analysis of Adrenal Gland Gene Expression in the 7-day-old Rat: Effects of Hypoxia from Birth

doi: 10.1152/physiolgenomics.00245.2006

Figure Lengend Snippet: Real-time PCR analysis of adrenal gene expression: verification of selected microarray results.

Article Snippet: Differences in gene expression, expressed as fold-change, were calculated using the 2 −ΔΔCt method using 18S as the internal control. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Symbol Assay ID# Cytochrome P450, subfamily 1B, polypeptide 1 Cyp1b1 Rn00564055_m1 Guanosine monophosphate reductase Gmpr Rn00589361_m1 Xanthine dehydrogenase Xdh Rn00567654_m1 Cadherin 1 Cdh1 Rn00580109_m1 Phospholipase A2, group IVA (cytosolic, Ca-dependent) Pla2g4a Rn00591916_m1 Aldehyde oxidase Aox1 Rn00571242_m1 Regulator of G-protein signaling protein 2 Rgs2 Rn00584932_m1 Carnitine palmitoyltransferase 2 Cpt2 Rn00563995_m1 Steroid 5 alpha-reductase 1 Srd5a1 Rn00567064_m1 Enolase 1, alpha Eno1 Rn00820594_g1 N-ethylmaleimide sensitive factor Nsf Rn00572694_m1 Cytochrome P450, subfamily 11B, polypeptide 3 Cyp11b3 Rn00822066_g1 Steroidogenic acute regulatory protein StAR Rn00580695_m1 Cytochrome P450, subfamily 11A Cyp11a Rn00568733_m1 Cytochrome P450, subfamily 21A, polypeptide 1 Cyp21a1 Rn00588996_g1 3-hydroxy-3-methylglutaryl-Coenzyme A reductase Hmgcr Rn00565598_m1 Low density lipoprotein receptor Ldlr Rn00598438_m1 Vascular endothelial growth factor Vegf Rn00582935_m1 Hypoxia-inducible factor 1 alpha Hif1a Rn00577560_m1 Open in a separate window Each Gene Expression Assay consisted of a mix of sequence specific forward/reverse primers and a FAM-labeled probe.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Microarray