Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 1. Schematic illustration of BMSCs-loaded porous particles fabrication. a. The generation of porous microcapsules with microfluidic electrospray; b. BMSCs- encapsulated microspheres intraperitoneally regulate the inflammatory microenvironment to alleviate liver function and fibrosis through paracrine and TGF- β/Smad signaling pathway for liver cirrhosis.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques:

Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 4. Therapeutic efficacy of Mi-BMSCs or BMSCs in TGF-β1-induced HSCs activation. (a) Cell viability of inactivated HSCs with different treatments at 24h, 48 h. (b–c) Total apoptosis of activated HSCs with different treatments at 24 h assayed by flow cytometry analysis (upper and lower right quadrants represent late and early apoptotic cells). (d–f) The relative mRNA levels of α-SMA, TGF-β, and IL-6 measured by qRT-PCR in Control, TGF-β1, BMSCs, Mi-BMSCs group. (g–l) Western blot and quantification analysis of COL1A1 (h), E-cadherin (i), N-cadherin (j), TGF-β R II (k) and TGF-β (l) in Control, TGF-β1, BMSCs, Mi-BMSCs group. Data are expressed as Means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs TGF-β1 group.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques: Drug discovery, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot

Journal: Materials today. Bio

Article Title: Mi-BMSCs alleviate inflammation and fibrosis in CCl 4 -and TAA-induced liver cirrhosis by inhibiting TGF-β/Smad signaling.

doi: 10.1016/j.mtbio.2024.100958

Figure Lengend Snippet: Fig. 10. The mechanism of Mi-BMSCs in the treatment of liver cirrhosis. (a–b) The representative fluorescent images and quantification of TGF-β from liver tissues of mice in Control, LF, BMSCs, Mi-BMSCs group by Immunofluorescent (IF) staining. Scale bar = 100 μm. (c) The serum levels of TGF-β in each group by ELISA. (d) The mRNA levels of TGF-β from liver tissues in each group by qRT-PCR assay. (e–h) The protein relative expression and quantification of TGF-β (g), the phosphorylation of Smad2 (f) and Smad2/3 (g) of liver tissues in Control, LF, BMSCs, Mi-BMSCs group by WB analysis *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs LF group; Data are expressed as Means ± SD.

Article Snippet: PCNA, IL-6, N-Cadherin, E-Cadherin, and TGF-β Fibrosis Pathway Antibody Sampler Kit antibodies were obtained from CST (USA).

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Phospho-proteomics

Journal: Journal of ovarian research

Article Title: IFN-γ in ovarian tumor microenvironment upregulates HLA-E expression and predicts a poor prognosis.

doi: 10.1186/s13048-023-01286-z

Figure Lengend Snippet: Fig. 3 IFN-γ-induced HLA-E overexpression was associated with JAK/ STAT1 signaling pathway. IFN-γ treatment activated JAK1/2 and STAT1, and HLA-E expression was not upregulated by IFN-γ in the presence of JAK1/2 inhibitor (50µM Ruxolitinib). Data shown represented one of three inde pendent experiments

Article Snippet: The primary antibodies were used as follows: HLA-E antibody (Abcam, MEM-E/02, 1:1000), Vinculin antibody (CST, #4650, 1:500), GAPDH antibody (Proteintech, 60004-1-Ig, 1:50000), IFN-γ signaling pathway antibody sampler kit containing antibodies of JAK1, p-JAK1, JAK2, p-JAK2, STAT1, p-STAT1 (CST, #44,902, 1:1000), which were incubated at 4 oC overnight.

Techniques: Over Expression, Expressing

Journal: Journal of ovarian research

Article Title: IFN-γ in ovarian tumor microenvironment upregulates HLA-E expression and predicts a poor prognosis.

doi: 10.1186/s13048-023-01286-z

Figure Lengend Snippet: Fig. 4 IFN-γ upregulated HLA-E expression through activation of STAT1 signal transduction pathway. (A) Caerulomycin A suppressed IFN-γ-induced STAT1 phosphorylation. HLA-E protein expression was also decreased with the increase of Caerulomycin A concentration. (B) Cell surface HLA-E expres sion was not downregulated by Caerulomycin A treatment. (C) SKOV3 cells transfected with EGFP were observed after being cultured with NKL cells for 4 h. More tumor cells were survived in IFN-γ stimulating group than in control group (without treatment). Caerulomycin A did not abrogate the IFN-γ- induced survivals. The bars = 250 μm. (D) IFN-γ reduced the function of NK lysis, and Caerulomycin A had no effect on IFN-γ-mediated killing inhibition, which was shown through LDH cytotoxicity assay kit. **P < 0.01, ***P < 0.001. Data shown represented one of three independent experiments

Article Snippet: The primary antibodies were used as follows: HLA-E antibody (Abcam, MEM-E/02, 1:1000), Vinculin antibody (CST, #4650, 1:500), GAPDH antibody (Proteintech, 60004-1-Ig, 1:50000), IFN-γ signaling pathway antibody sampler kit containing antibodies of JAK1, p-JAK1, JAK2, p-JAK2, STAT1, p-STAT1 (CST, #44,902, 1:1000), which were incubated at 4 oC overnight.

Techniques: Expressing, Activation Assay, Transduction, Phospho-proteomics, Concentration Assay, Transfection, Cell Culture, Control, Lysis, Inhibition, LDH Cytotoxicity Assay