Journal: The Journal of Clinical Investigation

Article Title: Mature T cell responses are controlled by microRNA-142

doi: 10.1172/JCI78753

Figure Lengend Snippet: (A) Impact of overexpression of atypical E2F proteins on cell cycle in WT T cells. WT T cells were infected with E2F7/E2F8 lentiviral particles, treated with anti-CD3 and anti-CD28 Abs, and analyzed for cell cycle. Combined data were from 3 independent experiments. (B) Overexpression of E2F7/E2F8 significantly inhibited T cell proliferation, as demonstrated by 3H-TdR incorporation. Data represent combination of 3 independent experiments. (C) Impact of E2F7/E2F8 silencing on cell cycle in miR-142 KO T cells. miR-142 KO T cells were targeted for E2F7/E2F8 silencing as described in Methods, then treated with anti-CD3 and anti-CD28 Abs for 0 to 3 days. Cell-cycle analyses were processed. Combined data were from 3 independent experiments. (D) KD of E2F7/E2F8 in miR-142 KO T cells significantly improved cell proliferation, as demonstrated by 3H-TdR incorporation. Data represent combination of 3 independent experiments. (E) Essential role of E2F7/E2F8 in cell-cycle activities in WT T cells. WT T cells were targeted for E2F7/E2F8 silencing, treated with anti-CD3 and anti-CD28 Abs for 0 to 4 days, and processed for cell-cycle analyses as in C. Data were combined from 3 independent experiments. (F) KD of E2F7/E2F8 in WT T cells significantly enhanced cell proliferation as demonstrated by 3H-TdR incorporation. Data represent combination of 3 independent experiments. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01, Holm-Sidak method (A, C, and E); unpaired t test (B, D, and F).

Article Snippet: Expression plasmids were cloned by inserting PCR products into the lentiviral U6-based expression vector (Addgene, 44248) digested by BstXI and XhoI.

Techniques: Over Expression, Infection

Journal: Advanced Science

Article Title: Targeting Transient Receptor Potential Melastatin‐2 (TRPM2) Enhances Therapeutic Efficacy of Third Generation EGFR Inhibitors against EGFR Mutant Lung Cancer

doi: 10.1002/advs.202310126

Figure Lengend Snippet: VDR is a predicted putative transcriptional factor within the TRPM2 5′‐flanking regulatory region (A), ranks top among genes suppressed by osimertinib in RNA‐seq analysis (B) and mediates TRPM2 downregulation by osimertinib in EGFRm NSCLC cells (C–F) via a VDRE present in the promoter region of TRPM2 (H–J) independent of c‐Myc (G). A , Putative transcriptional factors were predicted with PROMO program. B , Alterations in transcriptional factors in RNA‐seq data generated from both PC‐9 and HCC827 cells exposed to DMSO or 100 nM osimertinib (Osim) for 14 h are presented in the heatmap. C , Both PC‐9 and HCC827 cells were exposed to DMSO or 100 nM for the indicated times. D and G , The tested cell lines were transfected with the indicated siRNAs for 48 h. E , The indicated cell lines were infected with lentiviruses carrying VDR shRNA followed by puromycin selection. F , The indicated cell lines expressing vector (V) and VDR gene, respectively, were exposed to DMSO or 200 nM osimertinib for 16 h. After the aforementioned treatments, the proteins of interest were detected with Western blotting. H , Reporter constructs harboring the TRPM2 promoter region and deleted regions. I , The indicated cell lines were transfected with the given reporter constructs for 24 h followed with 200 nM osimertinib for another 16 h. Cells were then harvested for luciferase assay. The data are the means ± SD of four replicate determinations. Statistical differences were assessed with two‐sided unpaired Student's t ‐test. J , The indicated biotin‐oligos were incubated from whole‐cell protein lysates prepared from the indicated cell lines treated with DMSO or 200 nM osimertinib for 16 h. Pulldown assay was conducted with the streptavidin‐agarose beads followed by Western blotting to detect VDR bound to the oligos. Scr, scramble oligo; wt, VDRE‐wt oligo.

Article Snippet: TRPM2 siRNA (sc‐42674), c‐ Myc siRNA (sc‐29226), VDR siRNA (sc‐106692), and VDR shRNA plasmid (sc‐106692‐SH) were purchased from Santa Cruz Biotechnology.

Techniques: RNA Sequencing, Generated, Transfection, Infection, shRNA, Selection, Expressing, Plasmid Preparation, Western Blot, Construct, Luciferase, Incubation