pan pim kinase inhibitors pim447  (MedChemExpress)

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: Single-cell suspension from P14 TCR-transgenic mouse lymph nodes were activated with gp33 peptide (100 ng/mL), IL-2 (20 ng/mL), and IL-12 (2 ng/mL) for 2 days, then split daily into fresh media containing IL-2 (20 ng/mL). On day 5 of culture IL-2 expanded CTL were treated with pan-PIM kinase inhibitors PIM447 (5 µM) or AZD1208 (10 µM), or DMSO vehicle control for 24 hr and harvested on day 6 of culture to measure ( A ) Cell number ( B ) FSC-A SSC-A. Proteome analysis was also performed on inhibitor-treated CTL to measure ( C ) protein content, ( D ) protein expression of SCD1-3, SLC2A1, SLC2A3, GZMB, or ( F ) PDCD4. ( E ) Day 6 IL-2 expanded CTL from WT (C57BL/6) mice were treated for 24 hr with PIM447 or AZD1208 (both 1 µM) and GZMB expression was measured by flow cytometry. Symbols show biological replicates. Error bars show mean ± S.D. Data are representative of ( E ) n=2 biological replicates collected across at least two independent experiments. Proteomics analysis was performed on biological triplicates, with ( A, B ) collected in parallel with proteomics analysis. * q<0.05. Figure 4—figure supplement 2—source data 1. Raw values plotted in .

Article Snippet: Where indicated CTL were treated with 100 nM Tofacitinib (Selleckchem), 20 nM of rapamycin (Merck), 20 ng/mL PDBu (Cell Signaling Technologies) and 500 ng/mL ionomycin (Merck) or the pan PIM kinase inhibitors PIM447 (1 or 5 μM as indicated) and AZD1208 (1 or 10 μM as indicated; both Medchemexpress).

Techniques: Suspension, Transgenic Assay, Control, Expressing, Flow Cytometry

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: RNAseq analysis was performed in day 6 IL-2 expanded WT and Pim dKO CD8 T cells which were collected in parallel with proteomics analysis described in . ( A ) Volcano plot of RNAseq data, differentially expressed mRNA (FC >1.5, q<0.05) are highlighted in red. ( B ) Volcano plot of RNAseq data, Granzymes C-K, perforin, Pdcd4 and Sell are highlighted in red. ( C ) Heatmap of mRNA expression (TPM) for granzymes, perforin and effector cytokines. Bar chart of mRNA expression (TPM) of ( D ) Granzymes A and B ( E ) Glucose transporters Slc2a1 and Slc2a3. ( F, G ) Fold change of PimdKO/WT protein from proteomics analysis described in vs mRNA ( F ) highlighting in red proteins that are differentially expressed (FC >1.5, q<0.05) where mRNA is not substantially different (FC <1.2) and ( G ) highlighting in red protein and mRNA that are both differentially expressed (FC >1.5, q<0.05). ( H ) Estimated cytosolic ribosome content per cell (left), % ribosome of total cellular protein content (right). ( I ) Estimated protein copy number per cell of translation repressor PDCD4. ( J ) PDCD4 expression measured by flow cytometry on day 3 and 6 in IL-2 expanded WT vs Pim dKO CD8 T cells. ( K ) Estimated protein copy number per cell of EIF4A1. ( L ) Adjusted ratio of PDCD4: EIF4A1 (assuming 1 PDCD4 binds 2 x EIF4A1) in WT and Pim dKO proteomes. ( M ) Protein synthesis measured by OPP incorporation in day 6 IL-2-expanded WT CTL treated for 24 hours with pan PIM kinase inhibitors PIM447 (5 µM) or AZD1208 (10 µM). 30-min cycloheximide (100 µg/mL) treatment gives no protein synthesis background control. Symbols in bar charts show biological replicates: error bars show mean ± S.D. Data are representative of ( M ) n=2 biological replicates collected over two independent experiments, ( J ) n=2 biological replicates. Quantitative proteomics and RNAseq were performed on biological triplicates. * indicates q<0.05, fold-change (FC) shown on graph when q<0.05. Figure 5—source data 1. Raw values plotted in .

Article Snippet: Where indicated CTL were treated with 100 nM Tofacitinib (Selleckchem), 20 nM of rapamycin (Merck), 20 ng/mL PDBu (Cell Signaling Technologies) and 500 ng/mL ionomycin (Merck) or the pan PIM kinase inhibitors PIM447 (1 or 5 μM as indicated) and AZD1208 (1 or 10 μM as indicated; both Medchemexpress).

Techniques: Expressing, Flow Cytometry, Control, Quantitative Proteomics

Journal: Inflammation Research

Article Title: The role of Pim-1 kinases in inflammatory signaling pathways

doi: 10.1007/s00011-024-01924-2

Figure Lengend Snippet: The expression levels of Pim-1 and pro-inflammatory cytokines in LPS-stimulated macrophage-like THP-1 cells. ( A ) Cells were stimulated with LPS (0.01, 0.1, 1, and 2 µg/mL) for 6 h. Whole cell lysates were isolated and used to measure the protein expression levels of Pim-1, p-Bad, and pro-IL-1β by Western blotting. ( B ) Cells were stimulated with LPS (1 µg/mL) for the indicated time points. Whole cell lysates were isolated and used to measure the protein expression levels of Pim-1, p-Bad, and pro-IL-1β by Western blotting. ( C - F ) Cells were stimulated with LPS (0.01, 0.1, 1, and 2 µg/mL) for 6 h. Total RNA was extracted, and used to evaluate the mRNA expression levels of PIM1 , IL1B , TNFα , and IL6 by real-time qPCR (* p < 0.05, ** p < 0.01, # p < 0.001). ( G ) THP-1 cells were differentiated into macrophages using 100 nM PMA for 24 h, then the cells were stimulated with LPS (1 µg/mL) for 6 h after pre-treatment with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h. Whole cell lysates were isolated and used to measure the protein expression levels of IL-1β, TNF-α and IL-6 by Western blotting. ( H ) Whole cell lysates were isolated and used to measure the protein expression levels of Pim-1, p-Bad (Ser112), and Bad by Western blotting

Article Snippet: The pan-PIM kinase inhibitors, PIM447 and AZD1208, were purchased from Selleck Chemicals (#S7985, #S7104, Houston, TX, USA).

Techniques: Expressing, Isolation, Western Blot

Journal: Inflammation Research

Article Title: The role of Pim-1 kinases in inflammatory signaling pathways

doi: 10.1007/s00011-024-01924-2

Figure Lengend Snippet: The effect of PIM1 knockdown in LPS-induced iNOS, COX-2, MAPKs and NF-κB in macrophage-like THP-1 cells. ( A ) Whole cell lysates were isolated and used to measure the protein expression levels of iNOS and COX-2 by Western blotting. ( B , C ) Total RNA was extracted, and used to evaluate the mRNA expression levels of iNOS and COX-2, respectively (** p < 0.01, # p < 0.001). ( D ) Cells were stained with antibodies to COX-2 (red) and DAPI (blue) and captured at ×200 using fluorescence microscope (scale bar = 50 μm). ( E ) Cells were stimulated with LPS (1 µg/mL) for the indicated time points. Whole cell lysates were isolated and used to measure the protein expression levels of p-ERK, ERK, p-JNK, JNK, p-p38, and p38 by Western blot analysis. ( F ) Whole cell lysates were isolated and used to measure the protein expression levels of p-IKKα/β and p-NF-κB p65 by Western blotting. ( G ) Cells were stained with antibodies to NF-κB p65 (green) and DAPI (blue) and captured at ×200 using fluorescence microscope (scale bar = 50 μm). ( H ) Cytoplasmic and nuclear proteins were extracted and assayed by Western blot analysis using anti-NF-κB p65 antibody. The expression levels of actin and histone 3 were used as loading controls. ( I ) THP-1 cells were differentiated into macrophages using 100 nM PMA for 24 h, then the cells were stimulated with LPS (1 µg/mL) for 6 h after pre-treatment with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h. Whole cell lysates were isolated and used to measure the protein expression levels of p-IKKα/β and p-NF-κB p65 by Western blotting

Article Snippet: The pan-PIM kinase inhibitors, PIM447 and AZD1208, were purchased from Selleck Chemicals (#S7985, #S7104, Houston, TX, USA).

Techniques: Knockdown, Isolation, Expressing, Western Blot, Staining, Fluorescence, Microscopy

Journal: Inflammation Research

Article Title: The role of Pim-1 kinases in inflammatory signaling pathways

doi: 10.1007/s00011-024-01924-2

Figure Lengend Snippet: The effect of PIM1 knockdown in LPS-mediated activation of NLRP3 inflammasome in macrophage-like THP-1 cells. ( A ) Cells were stimulated with LPS (1 µg/mL) for the indicated time points. Whole cell lysates were isolated and used to measure the protein expression levels of NLRP3, ASC, pro-IL-1β, and pro-caspase-1 by Western blotting. ( B ) Cells were stimulated with LPS (1 µg/mL) and/or ATP (1 mM) for 6 h. Cell lysate (Lysate) and media supernatant (Sup) were isolated and used to measure the protein expression levels of pro-IL-1β, IL-1β, pro-caspase-1, and caspase-1 for the Sup as well as NLRP3, ASC, pro-caspase-1, and pro-IL-1β for the Lysate by Western blot analysis. ( C ) RAW 264.7 cells transfected with control siRNA or Pim-1 siRNA for 72 h and then stimulated with LPS (500 ng/mL) for 6 h. Whole cell lysates were isolated and used to measure the protein expression levels of NLRP3, caspase-11, pro-IL-1β, pro-caspase-1, and caspase-1 by Western blotting. ( D - F ) Total RNA was extracted, and used to evaluate the mRNA expression levels of NLRP3 , IL1B , and PIM1 , respectively (# p < 0.001). ( G , H ) Cells were differentiated into macrophages using 100 nM PMA for 24 h, then the cells were stimulated with LPS (1 µg/mL) with/without ATP (1mM) for 6 h after pretreatment with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h. Whole cell lysates were isolated and used to measure the protein expression levels of NLRP3, ASC, pro-IL-1β, and pro-Caspase-1 by Western blotting

Article Snippet: The pan-PIM kinase inhibitors, PIM447 and AZD1208, were purchased from Selleck Chemicals (#S7985, #S7104, Houston, TX, USA).

Techniques: Knockdown, Activation Assay, Isolation, Expressing, Western Blot, Transfection, Control

Journal: Inflammation Research

Article Title: The role of Pim-1 kinases in inflammatory signaling pathways

doi: 10.1007/s00011-024-01924-2

Figure Lengend Snippet: The effect of PIM1 knockdown in LPS-mediated activation of TAK1 expression in macrophage-like THP-1 cells. ( A ) Whole cell lysates were isolated and used to measure the protein expression levels of TLR4 and MyD88 by Western blotting. ( B ) Cells were stimulated with LPS (1 µg/mL) for 6 h after pre-treatment with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h. Whole cell lysates were isolated and used to measure the protein expression levels of TLR4 and MyD88 by Western blotting. ( C - E ) Cells were transfected with control siRNA or PIM1 siRNA, and pre-treatment with TAK1 inhibitor (5Z)-7-Oxozeaenol (1 µM), or with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h before LPS (1 µg/mL) stimulation. Whole cell lysates were isolated and used to measure the protein expression levels of p-TAK1, TAK1 and pro-IL-1β by Western blotting. Interaction of Pim-1 with TAK1 protein in LPS-induced THP-1 cells. ( F ) THP-1 cells were stimulated with LPS (1 µg/mL) for 6 h, and cell lysates were subjected to immunoprecipitation with TAK1, then the protein expression levels of Pim-1 and TAK1 were detected by Western blotting. ( G ) Cells were transfected with control siRNA or PIM1 siRNA, and Whole cell lysates were subjected to immunoprecipitation with TAK1, then the protein expression levels of Pim-1 and TAK1 were detected by Western blotting. ( H ) Cells were pre-treatment with 1 µM of TAK1 inhibitor (5Z)-7-Oxozeaenol for 1 h before LPS (1 µg/mL) stimulation. Whole cell lysates were subjected to immunoprecipitation with Pim-1, then the protein expression levels of TAK1 and Pim-1 were detected by Western blotting. ( I , J ) RAW 264.7 and BV2 cells were transiently transfected with plasmids expressing pMX-IRES-EGFP-Flag empty vector (control) or pMX-Pim-1 DN, pMX-Pim-1 WT, and pMX-Pim-1 K67M (mutant forms). The cell lysates were subjected to immunoprecipitation with anti-Flag, then the protein expression levels of TAK1 and Flag were detected by Western blotting

Article Snippet: The pan-PIM kinase inhibitors, PIM447 and AZD1208, were purchased from Selleck Chemicals (#S7985, #S7104, Houston, TX, USA).

Techniques: Knockdown, Activation Assay, Expressing, Isolation, Western Blot, Transfection, Control, Immunoprecipitation, Plasmid Preparation, Mutagenesis

Journal: Inflammation Research

Article Title: The role of Pim-1 kinases in inflammatory signaling pathways

doi: 10.1007/s00011-024-01924-2

Figure Lengend Snippet: The effect of PIM1 knockdown in LPS-induced JAK/STAT pathway in macrophage-like THP-1 cells. ( A ) Cells were transfected with control siRNA or PIM1 siRNA. ( B ) Cells were pre-treatment with TAK1 inhibitor (5Z)-7-Oxozeaenol (1 µM). ( C ) Cells were with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h before LPS (1 µg/mL) stimulation. Whole cell lysates were isolated and used to measure the protein expression levels of p-JAK1, JAK1, p-STAT3, STAT3, and pro-IL-1β by Western blotting

Article Snippet: The pan-PIM kinase inhibitors, PIM447 and AZD1208, were purchased from Selleck Chemicals (#S7985, #S7104, Houston, TX, USA).

Techniques: Knockdown, Transfection, Control, Isolation, Expressing, Western Blot