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: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 3: CUDC-101 inhibits ATC cell proliferation, and induces cell cycle arrest and apoptosis. (A) Basal expression of HDAC1, HDAC2 and EGFR in ATC cell lines. (B) Cell proliferation assay. Error bars are mean ± SD. (C) Cell cycle analysis after 24 hours of treatment. (D) The Caspase-Glo 3/7 assay after 48 hours of treatment with CUDC-101. *p < 0.05, **p < 0.01, ***p < 0.001. NS, no significant difference.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Proliferation Assay, Cell Cycle Assay, Caspase-Glo Assay

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 5: Protein targets of CUDC-101 in ATC cells. (A) CUDC-101 inhibits HDACs and MAPK in ATC cells. (B) Targets of CUDC-101 identified by phospho-kinase array. Phospho-kinase arrays were performed using cell lysates treated with and without CUDC-101. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. (C) Differentially expressed apoptotic proteins with and without CUDC-101 treatment. Apoptosis arrays were performed. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. For A–C, ATC cells were treated with the vehicle or CUDC-101 at 1.1 μM for 24 hours. (D) CUDC-101 reduces the expression of survivin, XIAP, and β-catenin. Cells were treated with the vehicle or CUDC-101 for 24 hours.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing

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: Frontiers in Cell and Developmental Biology

Article Title: SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells

doi: 10.3389/fcell.2022.854397

Figure Lengend Snippet: Dysregulation of the mTOR signaling pathway. (A) . Heat map representation of phosphoprotein ratio detected in full moon Biosystems mTOR phospho-protein array. Fold change was calculated after normalizing the average signal cor-responding to the median signal for each group, and the ratio has been calculated as KO vs. WT signals. Proteins that have shown at least 2-fold up or downregulation were included. (B) . KO cells show less co-localization of mTOR (green) with the lysosome (LAMP1, red) in refed and starved conditions than WT cortical cells. Scale bar represents 10 um. (C,D) The total intensity of mTOR and LAMP1 was measured with cell profiler using immunostaining images in WT (C) and (D) KO. Graphs represent mean ± SEM; * p < 0.05, ** p < 0.01, Student’s t-test ( n = 15–17 images for each condition). (E–G) Data representation of the total mean of intensity of (E) mTOR and (F) LAMP1 in an area of interest in the cytoplasm (doughnut) of WT cells at different conditions, respectively. Whispers max and min plot represent total mTOR and total LAMP1 intensity in the cell. (G) mTOR and LAMP1 correlation in basal, starved, and refed conditions. The graph rep-resents Pearson’s co-efficient correlation between LAMP1 and total mTOR in WT and KO cells. (H–L) Bar graph represents changed upstream protein phosphorylation changes at basal (H) , starved (J) , and refed (L) conditions. Further repre-sentation of phosphorylated target of downstream regulators altered at basal (I) , starved (K) , and refed conditions (M) . (N,O) Further targeted protein changed from the transition from basal to starved upstream regulators (N) and downstream regulators (O) . (I,K) represents affected proteins between trans ion from starved to refed, upstream regulators (P) and downstream regulators (Q) .

Article Snippet: Human mTOR signalling Phospho-Specific Antibody Microarray was purchased from Full moon Biosystems.

Techniques: Protein Array, Immunostaining, Phospho-proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells

doi: 10.3389/fcell.2022.854397

Figure Lengend Snippet: The response in mTOR pathway and amino acid regualtion following SLC38A10 knockout. Complete transcriptomic data and changed gene list subjected to IPA software analysis. (A) Regulation in the mTOR pathway in response to SLC38A10 knockout in Basal condition, blue symbols indicate down regulation and red symbols up regulation. (B) Heatmap describing changes in transcript levels from the mTOR pathway during Basal, Starved and Refed conditions. (C) Heatmap describing changes in transcript levels from the p70S6 pathway during Basal, Starved and Refed conditions. (D) Regulation in the mTOR pathway in response to SLC38A10 knockout in Starved condition, blue symbols indicate down regulation and red symbols up regulation. (E) Regulation in the mTOR pathway in response to SLC38A10 knockout in Refed condition, blue symbols indicate down regulation and red symbols up regulation. (F) Neurological diseases predicted by Path Designer, in response to SLC38A10 knockout.

Article Snippet: Human mTOR signalling Phospho-Specific Antibody Microarray was purchased from Full moon Biosystems.

Techniques: Knock-Out, Software

Journal: Cancer Research

Article Title: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer–Initiating Cells

doi: 10.1158/0008-5472.can-10-4660

Figure Lengend Snippet: Figure 1. STAT3 phosphorylation of ALDHþ/CD133þ subpopulation of colon cancer cells is higher than unseparated and the ALDH/CD133 subpopulations. A, ALDHþ/CD133þ and ALDH/CD133 subpopulations were separated from SW480, HCT116, and DLD-1 colon cancer cells by flow cytometry. Phosphorylation of STAT3 (Y705), ERK 1/2 (T202/Y204), phospho- independent STAT3, and GAPDH were detected by Western blot. B, representative examples of the expression of P-STAT3, ALDH1, and CD133 were shown by immunohistochemistry (IHC) using colon cancer tissue microarray slides. Negative/weak staining of P-STAT3 (Y705)/ALDH1/CD133 (a) and positive staining of P-STAT3 (Y705)/ALDH1/CD133 (b and c) tumor tissues were shown. The spots for P-STAT3 (Y705), ALDH1 and CD133 were from the matched tissues section from the same patient. The negative controls are stained with no antibody. C,colon cancer tissue microarray slides were double-stained with P-STAT3 (Y705) and ALDH1 using immunofluorescence (IF). ALDH1 high expression tumor cells (cytoplasm, green) also expressed phosphylated-STAT3 in nuclei (red). Scale bar: 10 mm.

Article Snippet: Proteins were transferred on to polyvinylidene difluoride membrane and were probed with a 1:1,000 dilution of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques: Phospho-proteomics, Cytometry, Western Blot, Expressing, Immunohistochemistry, Microarray, Staining

Journal: Cancer Research

Article Title: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer–Initiating Cells

doi: 10.1158/0008-5472.can-10-4660

Figure Lengend Snippet: Figure 2. STAT3 inhibitors LLL12, Stattic, and STAT3 shRNA inhibited STAT3 phosphorylation and STAT3 downstream genes expression in ALDHþ/CD133þ colon cancer– initiating cells. The ALDHþ/CD133þ

Article Snippet: Proteins were transferred on to polyvinylidene difluoride membrane and were probed with a 1:1,000 dilution of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques: shRNA, Phospho-proteomics, Expressing

Journal: Cancer Research

Article Title: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer–Initiating Cells

doi: 10.1158/0008-5472.can-10-4660

Figure Lengend Snippet: Figure 3. LLL12 (A), Stattic (B), and STAT3 shRNA (C) inhibited cell viability of SW480, HCT116, and DLD-1 colon cancer–initiating cells. The ALDHþ/CD133þ subpo- pulation of colon cancer cells was seeded in 96-well plates (3,000 cells per well) in triplicates in a serum- free MEBM. The following day, cancer-initiating cells were treated with LLL12, Stattic, CTL, or STAT3 shRNA. MTT assay was used to determine cell viability. D, Stattic and LLL12 inhibited tumorsphere formation of ALDHþ/CD133þ

Article Snippet: Proteins were transferred on to polyvinylidene difluoride membrane and were probed with a 1:1,000 dilution of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques: shRNA, MTT Assay

Journal: Cancer Research

Article Title: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer–Initiating Cells

doi: 10.1158/0008-5472.can-10-4660

Figure Lengend Snippet: Figure 4. A, LLL12 (5 mmol/L) and Stattic (10 mmol/L) decreased the percentage of ALDHþ/CD133þ subpopulation. However, 10 mmol/L doxorubicin or 5-Fu increased the percentage of ALDHþ/CD133þ colorectal cancer–initiating cells (, P < 0.05). B, ELISA assay showed that ALDHþ/CD133þ subpopulation secreted more IL-6 than ALDH/CD133 subpopulation of colon cancer cells. C, the expression of ALDH1, CD133, IL-6, IL-6R, GP130, IL-8 was detected by RT-PCR. (a) ALDHþ/CD133þ and ALDH/CD133 colon cancer cells were collected after sorting. The expression of IL-6, IL-6R, GP130, IL-8 were higher in ALDHþ/CD133þ cells than ALDH/CD133 cells; (b) the expression of ALDH1, CD133, IL-6, GP130, and IL-8 were lower after treated with LLL12 in ALDHþ/CD133þ colon cancers. D, after sorting, ALDHþ/CD133þ HCT116 and/or SW480 colon cancer–initiating cells were infected with CTL, IL-6, and/or STAT3 shRNA. (a) IL-6 shRNA decreased STAT3 phosphorylation of SW480 and HCT116 colorectal cancer–initiating cells in vitro; (b) IL-6 and STAT3 shRNA inhibited ALDHþ/CD133þ HCT116 cancer-initiating cell growth in vivo.

Article Snippet: Proteins were transferred on to polyvinylidene difluoride membrane and were probed with a 1:1,000 dilution of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, shRNA, Phospho-proteomics, In Vitro, In Vivo

Journal: Cancer Research

Article Title: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer–Initiating Cells

doi: 10.1158/0008-5472.can-10-4660

Figure Lengend Snippet: Figure 5. LLL12 suppressed tumor growth in mouse xenografts with SW480 or HCT116 colon cancer stem cells. The mice were given daily intraperitoneal dosages of 5 mg/kg LLL12 or DMSO vehicle. Tumor volume (A-a), tumor mass (A-b), and tumor weight (B) were reduced in all LLL12-treated mice compared with DMSO vehicle group. C, one representative sample from tumor tissues generated from SW480 or HCT116 colon cancer–initiating cells showing STAT3 but not ERK1/2 phosphorylation were also inhibited by LLL12 treatment. D, at the end of treatments, tumors tissues were dissociated to obtain a single-cell suspension and analyzed by flow cytometry. LLL12 reduced the percentage of ALDHþ/CD133þ

Article Snippet: Proteins were transferred on to polyvinylidene difluoride membrane and were probed with a 1:1,000 dilution of antibodies (Cell Signaling Technology) against phosphospecific STAT3 (Tyrosine 705; P-STAT3), phospho-independent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Techniques: Generated, Phospho-proteomics, Suspension, Cytometry

Journal: Biology

Article Title: N-WASP Attenuates Cell Proliferation and Migration through ERK2-Dependent Enhanced Expression of TXNIP

doi: 10.3390/biology11040582

Figure Lengend Snippet: FOXO1 expression is reduced in HSC-5 NW cells. ( A ) Individual duplicate arrays of phospho-Ser319 FOXO1 for the HSC-5 CN (red) and HSC-5 NW (green) cell lysate protein microarrays; the complete Kinex KAM-880 microarray is shown in . The average values of HSC-5 CN and HSC-5 NW cell phospho-Ser319 FOXO1 arrays are shown. ( B ) Representative Western blots of FOXO1 and GAPDH in HSC-5 CN and HSC-5 NW cells; n = 3. ( C ) Densitometric quantification of the FOXO1/GAPDH ratio in HSC-5 CN and HSC-5 NW cells normalized to HSC-5 CN cells. ( D ) Total RNA was extracted from HSC-5 CN and HSC-5 NW cells and reverse-transcribed to cDNA for quantifying FOXO1 expression relative to MRPL-27 using real-time PCR, normalized to HSC-5 CN cells. ( E ) Representative Western blots of FOXO1 and GAPDH in HSC-5 CN and HSC-5 NW cells treated with 10 µM MG132 or DMSO; n = 3. ( F ) Densitometric quantification of the FOXO1/GAPDH ratio in HSC-5 CN and HSC-5 NW cells treated as in ( E ) normalized to DMSO-treated HSC-5 CN cells. ( G ) Representative immunofluorescence images of HSC-5 CN and HSC-5 NW cells treated as in ( E ) stained for FOXO1 (green). Nuclei were counterstained with DAPI (blue). Scale bar represents 20 µm, n = 3. All values are the mean ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To identify the signaling pathways altered in HSC-5 NW cells, we used the Kinex KAM-880 microarray kit, which detects 518 pan-specific proteins and 359 phospho-specific proteins critical for cell signaling pathways.

Techniques: Expressing, Microarray, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining