Journal: International Journal of Molecular Sciences

Article Title: Rationale for Combining the BCL2 Inhibitor Venetoclax with the PI3K Inhibitor Bimiralisib in the Treatment of IDH2- and FLT3-Mutated Acute Myeloid Leukemia

doi: 10.3390/ijms232012587

Figure Lengend Snippet: IC50 values cell lines (μM).

Article Snippet: The BMI1 inhibitor PTC596 (HY-112041), the MCL1 inhibitor S63845 (HY-100741), the MEK inhibitor trametinib (HY-10999), the STAT3 inhibitor C-188-9 (HY-112288) and the PI3K inhibitor PQR309 (HY-12868) were purchased at MedChemExpress (Monmouth Junction, NJ, USA).

Techniques:

Journal: Cells

Article Title: CEP55 Promotes Cell Motility via JAK2–STAT3–MMPs Cascade in Hepatocellular Carcinoma

doi: 10.3390/cells7080099

Figure Lengend Snippet: CEP55 promotes MMP expression via activating JAK2–STAT3 signaling. ( A ) Huh-7 cells were pre-infected with the indicated CEP55 shRNA for 72 h, and then cells were incubated with or without 20 ng/mL IL-6 for 30 min, followed by an analysis of the expression of the indicated proteins by western blotting; ( B ) Flag-tagged CEP55 was pre-transfected into Hep3B cells for 72 h, and then cells were incubated with or without 20 ng/mL IL-6 for 30 min, followed by an analysis of the expression of the indicated proteins by western blotting; ( C ) Vector or Flag-CEP55 plasmids were transfected into HepG2 cells. JAK2 was immunoprecipitated with anti-flag antibody; ( D ) Vector or flag-CEP55 plasmids were transfected into Hep3B cells. Ectopic CEP55 was immunoprecipitated with anti-JAK2 antibody; ( E ) Immunoprecipitation of CEP55, JAK2, and IgG in HepG2 cells, and western blotting analysis of the indicated proteins; ( F ) Flag-tagged CEP55 was pre-transfected into Hep3B cells for 24 h, followed by treatment with the control, JAK2, and STAT3 shRNA for 48 h. Expression levels of the indicated proteins were analyzed by western blotting.

Article Snippet: JAK2 inhibitor XL019 (#S7036), STAT3 inhibitor C188-9 (#S8605) was from Selleck (Shanghai, China).

Techniques: Expressing, Infection, shRNA, Incubation, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Cells

Article Title: CEP55 Promotes Cell Motility via JAK2–STAT3–MMPs Cascade in Hepatocellular Carcinoma

doi: 10.3390/cells7080099

Figure Lengend Snippet: Knockdown of JAK2 or STAT3 expression abrogates the pro-migration and -invasion effects on CEP55 overexpression. ( A ) Hep3B cells were pre-infected with flag-CEP55 for 24 h, and then were infected with indicated shRNA for 48 h. Cells’ migration ability was determined by wound healing assays. Representative microscope images are plotted. Bars, 50 μm; ( B ) A plot of the summary of the relative migration distance. The control group was set as 1. N = 4, * p < 0.005; ** p < 0.001; ( C ) Hep3B cells were pre-infected with flag-CEP55 for 24 h and were then infected with the indicated shRNA for 48 h. Cells’ invasion ability was determined by trans-well assays. Representative microscope images are plotted. Bars, 50 μm; ( D ) A plot of the summary of the relative invasion cell number. The control group was set as 1. N = 4, * p < 0.005; ** p < 0.001; ( E ) Hep3B cells were pre-infected with flag-CEP55 for 24 h and were then infected with 10 nM JAK2 inhibitor XL019 or 10 nM STAT3 inhibitor C188-9 for 48 h. Cells’ invasion ability was determined by trans-well assays. Representative microscope images are plotted. Bars, 50 μm; ( F ) A plot of the summary of the relative invasion cell number. The control group was set as 1. N = 4, ** p < 0.001.

Article Snippet: JAK2 inhibitor XL019 (#S7036), STAT3 inhibitor C188-9 (#S8605) was from Selleck (Shanghai, China).

Techniques: Knockdown, Expressing, Migration, Over Expression, Infection, shRNA, Microscopy, Control

Journal: Cells

Article Title: CEP55 Promotes Cell Motility via JAK2–STAT3–MMPs Cascade in Hepatocellular Carcinoma

doi: 10.3390/cells7080099

Figure Lengend Snippet: Model of CEP55 in JAK2–STAT3 signaling. CEP55 physiologically interacts with JAK2, leading to the heightening of JAK2 phosphorylation and activation induced by ligands, which activates JAK2 and then phosphorylates STAT3 which transfers into the nucleus, switching on the expression of MMP2/9 genes. Finally, CEP55 promotes the cell migration and invasion of HCC cells.

Article Snippet: JAK2 inhibitor XL019 (#S7036), STAT3 inhibitor C188-9 (#S8605) was from Selleck (Shanghai, China).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Migration

Journal: bioRxiv

Article Title: PD-L1 blockade restores CAR T cell activity through IFNγ-regulation of CD163+ macrophages

doi: 10.1101/2022.01.25.477150

Figure Lengend Snippet: (a-c) PD-L1 expression in macrophages and DU145-PSCA tumor cells in the immune-suppression assay. (d, e) Immunostaining of CD68 and PD-L1 in a humanized MISTRG mouse model following CAR T cell therapy against subcutaneous DU145-PSCA (d) and intratibial LAPC9 (e) prostate xenografts. (f, g) PD-L1 induction at the protein (f) and mRNA (g) levels following inhibition of IFNγ signaling. Anti-IFNγR1 antibody was used to block IFNγ signaling in the presence of recombinant IFNγ or CAR T cell-derived CM collected from the tumor cell killing assay. (h) PD-L1 induction following inhibition of various signaling pathways. CAR T cell-derived CM was applied to M2 macrophages in the presence of various small molecule inhibitors: fludarabine (STAT1 i), C188-9 (STAT3 i), itacitinib (JAK1 i), AG490 (JAK2 i), AZD1480 (JAK1/2 i), Bay11-7082 (NFκB i), Akti VIII (AKT i), CZC24832 (PI3K i), rapamycin (mTOR i). PD-L1 induction was evaluated by flow cytometry 48 hours after CM stimulation.

Article Snippet: Small molecule inhibitors included Fludarabine (STAT1 inhibitor, EnzoALX-480-100-M005), AZD1480 (JAK1 & JAK2 inhibitor, MilliporeSigma, SML1505-5MG), Itacitinib (JAK1 inhibitor, Cayman Chemicals, 27597), Rapamycin (mTOR inhibitor, Cayman Chemicals, 13346), C188-9 (STAT3 inhibitor, Cayman Chemicals, 30928), Akt Inhibitor VIII (AKT inhibitor, MilliporeSigma, 124018-5MG), BAY 11-7082 (NF-κB inhibitor, MilliporeSigma, B5556-10MG), AG490 (JAK2 inhibitor, MilliporeSigma, 658401-5MG), CZC24832 (PI3Kγ inhibitor, MilliporeSigma, SML1214-5MG).

Techniques: Expressing, Suppression Assay, Immunostaining, Inhibition, Blocking Assay, Recombinant, Derivative Assay, Flow Cytometry

Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells

doi: 10.3390/ijms21134584

Figure Lengend Snippet: IR induces RIP1 expression and activates epithelial-mesenchymal transition (EMT) in vitro/in vivo. ( A ) Lysates from A549 cells treated with IR (10 Gy) were subjected to immunoblotting to examine the expression levels of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3 p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( B ) A549 cells were exposed to IR (10 Gy) and treated with gefitinib (EGFR inhibitor, 10 µM). Immunoblotting was applied to examine the expression of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3, p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( C , D ) A549 cells subjected to IR (10 Gy) were treated with Src (20 µM) or STAT3 inhibitor (20 µM) for the indicated periods and Western blot analyses for RIP1, vimentin, MMP-2, MMP-9, Src, p-Src, STAT3, p-STAT3 and β-actin (loading control) were conducted. Data are representative of triplicate experiments. (***; p < 0.001) ( E ) A549-tumor xenografts in response to sham irradiation or irradiation with 10 Gy. Tumors were harvested 48 h after irradiation. Immunohistochemical (IHC) analysis of xenograft tissues of mice after irradiation was performed with antibodies against p-STAT3, STAT3 and Src. Hematoxylin and eosin (H&E) staining of xenograft tissues in mice after irradiation with 10Gy. Scale bar indicates 300 μm. ( F – H ) The correlation plot of IHC-score quantification for p-STAT3, STAT3 and Src. The IHC scores for p-STAT3, STAT3 are shown in ( F , G ), respectively. The IHC score for Src is shown in ( H ).

Article Snippet: Necrostatin (RIP1 inhibitor) and PP2 (Src inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA), C188-9 (STAT3 inhibitor III) from EMD Millipore Corp. (Billerica, MA, USA) and gefitinib (EGFR inhibitor) and BAY 11-7082 (NF-κB inhibitor) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Irradiation, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells

doi: 10.3390/ijms21134584

Figure Lengend Snippet: IR-mediated activation of EMT involves RIP1 kinase activity. ( A ) To determine the mechanism underlying IR-induced invasion of A549 cells, necrostatin was added to the lower chamber of a transwell system and cells were seeded onto the upper chamber. After 24 h, cells on the membrane were counted. (***; p < 0.001) ( B ) IR-induced migration of A549 cells. Experiments were performed as described in Materials and Methods. Representative data from triplicate experiments are shown on the right panel. (***; p < 0.001) ( C ) Cells were subjected to IR (10 Gy) and treated with necrostatin (20 µm), and apoptotic cell death determined via the PI uptake assay. Representative data from triplicate experiments are shown. ( D , E ) Immunoblotting for RIP1, MMP-2, MMP-9 EGFR, p-EGFR, Src, p-Src, STAT3 p-STAT3, vimentin and β-actin (loading control). ( F ) Immunofluorescence staining of RIP1-positive A549 cells after treatment with IR. Endogenous RIP1 expression in IR-treated or non-treated A549 cells was determined via immunofluorescence staining. A549 cells were stained with DAPI to visualize nuclei (blue) and immunolabeled with an anti-RIP1 antibody, which was detected with FITC-conjugated IgG (green). Scale bar: 10 μm.

Article Snippet: Necrostatin (RIP1 inhibitor) and PP2 (Src inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA), C188-9 (STAT3 inhibitor III) from EMD Millipore Corp. (Billerica, MA, USA) and gefitinib (EGFR inhibitor) and BAY 11-7082 (NF-κB inhibitor) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activation Assay, Activity Assay, Migration, Western Blot, Immunofluorescence, Staining, Expressing, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells

doi: 10.3390/ijms21134584

Figure Lengend Snippet: IR induces invasion/migration by promoting EMT via RIP1 activation. ( A , B ) A549 cells transfected with control or RIP1 siRNA for 24 h were subjected to invasion and migration assays. (***; p < 0.001) ( C , D ) Lysates from A549 cells transfected with control or RIP1 siRNA for 24 h were subjected to Western blot analysis for RIP1, MMP-2, MMP-9 EGFR, p-EGFR, Src, p-Src, STAT3 p-STAT3, vimentin and β-actin (loading control). ( E ) Immunofluorescence staining of RIP1-positive A549 cells after IR exposure. Endogenous RIP1 expression in IR treated or non-treated A549 cells was determined via immunofluorescence staining. A549 cells were stained with DAPI to visualize nuclei (blue) and immunolabeled with an anti-RIP1 antibody, which was detected with FITC-conjugated IgG (green). Scale bar: 10 μm.

Article Snippet: Necrostatin (RIP1 inhibitor) and PP2 (Src inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA), C188-9 (STAT3 inhibitor III) from EMD Millipore Corp. (Billerica, MA, USA) and gefitinib (EGFR inhibitor) and BAY 11-7082 (NF-κB inhibitor) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Activation Assay, Transfection, Western Blot, Immunofluorescence, Staining, Expressing, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells

doi: 10.3390/ijms21134584

Figure Lengend Snippet: Scheme of the potential IR-induced EGFR/NF-κB–RIP1–Src–STAT3–EMT pathway.

Article Snippet: Necrostatin (RIP1 inhibitor) and PP2 (Src inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA), C188-9 (STAT3 inhibitor III) from EMD Millipore Corp. (Billerica, MA, USA) and gefitinib (EGFR inhibitor) and BAY 11-7082 (NF-κB inhibitor) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: