Journal: Journal of Biological Chemistry

Article Title: Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation

doi: 10.1074/jbc.m112.361709

Figure Lengend Snippet: FIGURE 1. Expression of PIM kinases in Th cells is regulated by the knockdown of STAT4 and STAT6. A, expression of PIM1, PIM2, and PIM3 during human Th cell differentiation. Naïve CD4 Thp cells isolated from cord blood were activated (Th0) or stimulated with IL-12 (Th1) or IL-4 (Th2). Samples were harvested for Western blotting at the indicated time points. The panels show representative data from four biological replicates. B, expression of PIM1, PIM2, and PIM3 in activated human peripheral CXCR3 and CXCR3 CD4 T cells. CD4 cells were isolated from buffy coats, and the CXCR3-positive and -negative populations wereisolatedbyflowcytometriccellsorting.Sortedcellswereactivatedandharvestedattheindicatedtimepoints.Thepanelsshowrepresentativedataoffive biological replicates. C, CD4 T cells were nucleofected with STAT4 (pooled siRNAs 1 2) or NT siRNA and polarized in the Th1 direction 20–24 h after nucleofection. Samples were harvested for Western blotting at the indicated time points. The panels show representative data from three biological replicates. D, CD4 T cells were nucleofected with STAT6 or NT siRNA and polarized in the Th2 direction 24 h after nucleofection. Samples were harvested for Western blotting at the indicated time points. The panels show representative data from two biological replicates. Vertical lines represent repositioned gel lanes that are from the same blot and the same exposure. d, days.

Article Snippet: Proteins were detected using the following antibodies: mouse -PIM1 (12H8, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit -PIM2 (ATLAS Antibodies, Stockholm, Sweden or D1D2, Cell Signaling Technology, Beverly, MA), rabbit -PIM3 (D17C9, Cell Signaling Technology), mouse -T-BET (4B10, Santa Cruz Biotechnology), rabbit -STAT1 p84/p91 (E-23, Santa Cruz Biotechnology), rabbit -STAT4 (C-20, Santa Cruz Biotechnology), mouse -STAT6 (BD Biosciences), mouse -GAPDH (number 5G4, 6C5, HyTest, Turku, Finland), and mouse - -actin (Sigma-Aldrich).

Techniques: Expressing, Knockdown, Cell Differentiation, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation

doi: 10.1074/jbc.m112.361709

Figure Lengend Snippet: FIGURE 2. Knockdown of PIM kinases down-regulates IFN at mRNA and protein levels during Th1 cell polarization. A, PIM1, PIM2, and PIM3 siRNAs efficiently inhibit the induction of PIM kinase expression in human CD4 cells. Naïve CD4 cells nucleofected with the indicated siRNAs were activated and polarized in the Th1 direction 20 h after nucleofection. Samples were harvested for Western blotting at the indicated time points. Representative data are shown from seven independent experiments. B, knockdown of PIM kinases inhibits IFN protein production. Cell culture supernatants from nucleofected and Th1-polarized cells were collected at the indicated time points, and the amount of IFN produced by the cells was measured by a cytokine assay. The bars represent the mean values S.E. (error bars) of the relative levels of IFN in PIM123 siRNA samples relative to the control samples. The values of the control samples were set as 1. Data collected from four to five independent experiments are shown. C, the induction of IFN mRNA is inhibited by PIM knockdown during early Th1 polarization. The levels of IFN mRNA in the nucleofected cells were analyzed by TaqMan RT-PCR. The graph represents the average -fold differences S.E. (error bars) of IFN mRNA levels in siRNA-treated Th1 cells compared with the average Thp value. D, PIM123 siRNA- or NT siRNA-nucleofected cells were cultured for 6 days under Th1-polarizing conditions. The cells where then harvested, and half of the cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin, whereas the other half was left unstimulated. Cells were stained for intracellular IFN and analyzed with flow cytometry. Bars represent the average S.E. (error bars) geometrical (Geo) mean of fluorescence intensity calculated from four independent experiments. Vertical lines repre- sent repositioned gel lanes that are from the same blot and the same exposure. Statistical significances were calculated using the two-tailed paired t test: *, p 0.05; **, p 0.02; ***, p 0.01. siRNAs used were NT siRNA or PIM123 siRNAs.

Article Snippet: Proteins were detected using the following antibodies: mouse -PIM1 (12H8, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit -PIM2 (ATLAS Antibodies, Stockholm, Sweden or D1D2, Cell Signaling Technology, Beverly, MA), rabbit -PIM3 (D17C9, Cell Signaling Technology), mouse -T-BET (4B10, Santa Cruz Biotechnology), rabbit -STAT1 p84/p91 (E-23, Santa Cruz Biotechnology), rabbit -STAT4 (C-20, Santa Cruz Biotechnology), mouse -STAT6 (BD Biosciences), mouse -GAPDH (number 5G4, 6C5, HyTest, Turku, Finland), and mouse - -actin (Sigma-Aldrich).

Techniques: Knockdown, Expressing, Western Blot, Cell Culture, Produced, Cytokine Assay, Control, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Cell reports

Article Title: The Neonatal and Adult Human Testis Defined at the Single-Cell Level

doi: 10.1016/j.celrep.2019.01.045

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Polyclonal antibody against PIM2 , GeneTex , Cat # GTX113928; RRID:AB_2037694.

Techniques: cDNA Synthesis, SYBR Green Assay, Software, Quantitative Proteomics

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 4 The effect of MXJDH on macrophage polarization and Pim2/NF-κB pathway. (A) Flow cytometry analysis of the effect of MXJDH on CD86 and CD206 expression in LPS-treated macrophages. (B) Co-IP analysis of the formation of Pim2-p65. (C and D) Effect of MXJDH and Dexamethasone on inflammatory cytokines TNF-α and IL-10 in LPS-treated macrophages (n=3). (E) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α. (F–J) Statistical analysis of p65, Pim2, IL-6, IL-10 and TNF-α protein levels (n=3). Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, VS model group.

Article Snippet: Reagents NF-κB-p65 Antibody (Cat No. 21014), NF-κB p65 Mouse Monoclonal Antibody (Cat No. 38054), PIM2 Rabbit mAb (Cat No. 56914), IL10 Antibody (Cat No. 38392), and goat anti-rabbit IgG Secondary Antibody (Cat No. L3012) were purchased from Signalway Antibodies (Jiangsu, China).

Techniques: Flow Cytometry, Expressing, Co-Immunoprecipitation Assay

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 5 MXJDH ameliorates sepsis by inhibiting the Pim2/NF-κB pathway. (A–E) Statistical analysis of mRNA expression levels of p65, Pim2, IL-6, IL-10 and TNF-α (n=3). (F–K) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α and statistical analysis (n=3). (L) Protein expression levels of p65 and Pim2 in cytoplasm and nucleus. (M) Representative images of P65, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 20 µm as indicated. Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, ****P < 0.0001, VS model group.

Article Snippet: Reagents NF-κB-p65 Antibody (Cat No. 21014), NF-κB p65 Mouse Monoclonal Antibody (Cat No. 38054), PIM2 Rabbit mAb (Cat No. 56914), IL10 Antibody (Cat No. 38392), and goat anti-rabbit IgG Secondary Antibody (Cat No. L3012) were purchased from Signalway Antibodies (Jiangsu, China).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 6 The effect of MXJDH on macrophages in sepsis mice. (A) Representative images of F4/80, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 25 µm as indicated. (B) Representative images of CD86, CD206 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 100 µm as indicated.

Article Snippet: Reagents NF-κB-p65 Antibody (Cat No. 21014), NF-κB p65 Mouse Monoclonal Antibody (Cat No. 38054), PIM2 Rabbit mAb (Cat No. 56914), IL10 Antibody (Cat No. 38392), and goat anti-rabbit IgG Secondary Antibody (Cat No. L3012) were purchased from Signalway Antibodies (Jiangsu, China).

Techniques: Immunofluorescence, Staining