Journal: Cell Reports

Article Title: PIM kinases regulate early human Th17 cell differentiation

doi: 10.1016/j.celrep.2023.113469

Figure Lengend Snippet: PIMs are upregulated in Th17 cells via the IL6/STAT3 axis (A) Reads per kilobase of transcript, per million mapped reads (Rpkm) of PIM1 , PIM2 , and PIM3 are depicted at different times of activation (Th0) or Th17 differentiation from three biological replicates, using our published RNA-seq data (GEO: GSE52260). (B) The expression of the three PIMs in Th0 and Th17-polarizing cells over time was analyzed by western blot (bottom). Band intensities of target proteins from four biological replicates were normalized to β-actin (top). (C) Representative western blots of PIM1, PIM2, and PIM3 are shown from naive CD4 + T cells cultured for 72 h under activated Th0 condition, Th17 differentiation, or activated Th0 in the presence of Th17 cytokines (IL6, IL1β, and TGFβ) (right). Graphs on the left show band intensities of target proteins from four biological replicates, normalized to β-actin and relative to Th0. Statistical significance was calculated by comparing each condition to Th0. (D) Western blots of STAT3, PIM1, PIM2, and PIM3 protein levels in non-targeting (Scr) vs. STAT3 KD cells, at 72 h of Th17 polarization are shown (left). Protein intensities of STAT3 and PIM kinases from three biological replicates were normalized to β-actin and relative to Scr (right). Graphs in (B)–(D) show mean ± SEM. Statistical significance was calculated using two-tailed Student’s t test ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: rabbit polyclonal anti-human PIM1 , Cell Signaling Tech , Cat# 2907; RRID: AB_2283785.

Techniques: Activation Assay, RNA Sequencing, Expressing, Western Blot, Cell Culture, Two Tailed Test

Journal: Cell Reports

Article Title: PIM kinases regulate early human Th17 cell differentiation

doi: 10.1016/j.celrep.2023.113469

Figure Lengend Snippet: PIMs negatively regulate expression of IL17 and RORC (A) Workflow. Naive CD4 + T cells were simultaneously transfected with a pool of one LNA and two siRNAs each targeting PIM1, PIM2, and PIM3, respectively, (TKD), or with in vitro transcribed PIM1, PIM2, and PIM3 RNAs (TOE). After 24 h resting, cells were cultured under Th17 conditions for 72 h. (B and C) PIM TKD efficiency was confirmed at RNA level at 6 h, 24 h, and 72 h post-differentiation in four biological replicates using qRT-PCR (B) or at protein level at 72 h of Th17 cell differentiation by western blot (C, left). Band intensities of PIM kinases from five biological replicates were normalized to β-actin and relative to Scr control (C, right). (D) Secreted IL17A cytokine levels in supernatants of PIM TKD Th17 cells are shown at 72 h of polarization. Boxplot represents median and interquartile range, and whiskers extend to maximum and minimum values. Data represent five biological replicates. (E–G) IL17 A/F RNA levels at 72 h (E and F) and RORC RNA levels at 48 and 72 h (G) in PIM TKD Th17 cells were analyzed in four biological replicates using qRT-PCR. (H–L) PIM overexpression was confirmed at 48 h of polarization by western blot (H, left). Band intensities of PIM kinases from three biological replicates were normalized to β-actin and relative to GFP control (H, right). (I–L) IL17 secretion (I), IL17A , and RORC RNA expression (J and K) and CCR6 surface expression (L) in PIM TOE Th17 cells at 72 h of polarization, were assessed by ELISA, qRT-PCR, and flow cytometry analyses, respectively, for three biological replicates. (L) Mean fluorescence intensity (MFI) values were normalized to Scr control. ELISA values in plots (D) and (I) were normalized for cell count (live), and then normalized to Scr or GFP control, respectively. (B), (E), (F), (G), (J), and (K) depict transcript FC normalized to control. Plots in (B, C, E–L) show mean ± SEM. Statistical significance is calculated using two-tailed Student’s t -test (ns not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). See also Figure S2 .

Article Snippet: rabbit polyclonal anti-human PIM1 , Cell Signaling Tech , Cat# 2907; RRID: AB_2283785.

Techniques: Expressing, Transfection, In Vitro, Cell Culture, Quantitative RT-PCR, Cell Differentiation, Western Blot, Control, Over Expression, RNA Expression, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Cell Counting, Two Tailed Test

Journal: Cell Reports

Article Title: PIM kinases regulate early human Th17 cell differentiation

doi: 10.1016/j.celrep.2023.113469

Figure Lengend Snippet: PIMs alters Th17 gene expression (A) Z score heatmaps standardized with a FDR of <0.1 and FC of >1.4 for the differentially expressed genes detected at 6 h and 24 h are shown for four biological replicates. Genes common between the two time points are highlighted in bold. (B) Venn diagram demonstrating the number of overlapping differentially expressed genes in PIM TKD Th17 cells at 6 h and 24 h of polarization with a FDR of <0.1 and a FC of >1.4. (C) IPA was used to identify signaling pathways that are significantly altered upon PIM TKD. For analysis, differentially expressed genes at 6 h and 24 h were merged and the top enriched pathways related to T cell signaling and immune-mediated diseases are shown. (D) Volcano plots highlight the Th17-associated transcripts that are differentially expressed upon co-depletion of PIM1, PIM2 and PIM3, at 6 h (left) and 24 h (right) of Th17 polarization with a FDR of <0.1 and a FC of >1.4. Upregulated genes are in pink, and downregulated genes are in blue. Genes colored in gray are selected by a FDR of <0.25. (E and F) RORA (E) and STAT 3 (F) gene expression was analyzed in PIM-depleted Th17 cells at 6 h and 24 h by qRT-PCR. FC normalized to the Scr control was plotted for four biological replicates. Boxplots represent median and interquartile range, and whiskers extend to maximum and minimum values. Statistical significance is calculated using two-tailed Student’s t tests ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). See also Figure S3 and Table S1 .

Article Snippet: rabbit polyclonal anti-human PIM1 , Cell Signaling Tech , Cat# 2907; RRID: AB_2283785.

Techniques: Gene Expression, Protein-Protein interactions, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Cell Reports

Article Title: PIM kinases regulate early human Th17 cell differentiation

doi: 10.1016/j.celrep.2023.113469

Figure Lengend Snippet:

Article Snippet: rabbit polyclonal anti-human PIM1 , Cell Signaling Tech , Cat# 2907; RRID: AB_2283785.

Techniques: Recombinant, Sequencing, Purification, Isolation, Enzyme-linked Immunosorbent Assay, DC Protein Assay, Gene Expression, Reverse Transcription, Staining, CRISPR, Plasmid Preparation, Software