Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A OWCM-155 NEPC organoid cells (5000/well) were plated with or without stable H19 knockdown (Lv-shH19 or Lv-Scr). Line graphs demonstrate the quantification of organoid growth. B Trp53 flox/flox /Rb1 flox/flox mouse organoid cells (5000/well/condition) were plated. Bar plot represents organoid area quantification ( n = 10; biological replicates) C Representative fluorescence images of organoids in Fig. 4B. Scale bar: 1000 μm. D Time course of OWCM-155 control (shScr) and H19 KD (shH19) tumor xenograft growth in n = 5 NSG mice. E Mean tumor weight of the tumor xenografts ( n = 4) harvested at 11-week post-injection. F Relative mRNA expression of H19 and NE markers in OWCM-155 shH19 vs. OWCM-155 shScr xenografts. Each bar represents pooled data from tumors from three different mice per group. G Organoid invasion assay. Transwell assay using FluoroBlock inserts with 50,000 cells/well of Trp53 flox/flox / Rb1 flox/flox organoids transduced with EV-GFP, Cre-GFP, and Cre-GFP + shH19 ( n = 3). Quantification by fluorescence intensity of the migrated cells on the bottom of the transwell, five days post-plating. EV-GFP was used as a control. H Growth response (left) of LNCaP cells with WT and P53/RB1 knockdown (shP53/RB1) with and without H19 knockdown (shH19 vs. Scr) treated with ENZA (5 µM) for 5 days. DMSO was used for the control treatment. WB (right) of these cells shows the RB and P53 knockout. I WB of LNCaP shP53/Rb1 cells with and without two different shH19 (shH19-C, shH19-D) treated with ENZA (2, 5 µM). DMSO was used as a control. J , Growth response of LNCaP cells overexpressing H19 vs. empty vector (EV) treated with ENZA (2, 5 µM) for 5 days. DMSO was a control treatment at 0 μM. K Western blot of LNCaP cells used in ( J ) treated with ENZA (2, 5 µM; 72 h). In H , I , and K , WB images were quantified using ImageJ (Methods) with Actin as a control. Data are mean ± SD ( D , G , H ), or mean ± SEM ( A , B , E , F , J ); n = 3 ( F – H , J ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test ( A , B , D – G , J ) or Tukey’s multiple comparisons test ( h ).

Article Snippet: For H19 knockdown in mouse organoids, plasmid GIPZ Mouse H19 shRNA purchased from Dharmacon (RMM4431), and for Cre recombinase expression, the plasmid FUGW-Cre (Addgene) was kindly provided by Dr. Owen Witte (UCLA, Los Angeles, CA). shOCT4 (LL-hOCT4i-1) (Addgene plasmid # 12198; http://n2t.net/addgene:12198 ; RRID:Addgene_12198) and shNANOG (LL–hNANOGi) were a gift of George Daley (Addgene plasmid # 12196; http://n2t.net/addgene:12196 ; RRID:Addgene_12196) . shSOX2 (pLKO.1 Sox2 HM) was a gift from Matthew Meyerson (Addgene plasmid # 26353; http://n2t.net/addgene:26353 ; RRID:Addgene_26353) . shP53 (pLKO-p53-shRNA-941) (Addgene plasmid # 25637; http://n2t.net/addgene:25637 ; RRID:Addgene_25637) and shRB1 (pLKO-RB1-shRNA19) (Addgene plasmid# 25640; http://n2t.net/addgene:25640 ; RRID:Addgene_25640) , were a gift from Todd Waldman.

Techniques: Knockdown, Fluorescence, Control, Injection, Expressing, Invasion Assay, Transwell Assay, Transduction, Knock-Out, Plasmid Preparation, Western Blot, Two Tailed Test

Journal: Oncotarget

Article Title: Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

doi: 10.18632/oncotarget.2500

Figure Lengend Snippet: Figure 5: Rac1 inhibition abolishes IR-induced activation of both ATM and ATR signaling pathways. CD18/HPAF cells were treated with/without 10 Gy IR in the presence of NSC23766 at the indicated doses and incubated for 1 h at 37oC. (A) To assess ATR and ATM kinase activities, ATR and ATM were immunoprecipitated from the cell lysates using anti-ATR (N-19) and anti-ATM (2C1) antibodies respectively and assayed for relative kinase activity using recombinant p53 protein as substrate. (B) To measure Chk1 and Chk2 activity, Chk1 and Chk2 were immunoprecipitated from the cell lysates using anti-Chk1 (G-4) and anti-Chk2 (B-4) antibodies respectively and assayed for relative kinase activity using recombinant Cdc25C protein as substrate. As controls, protein levels of ATR, ATM, Chk1 and Chk2 in the immunoprecipitates (IP-WB) as well as in the cell lysates (WB) were assessed by immunoblotting. (C) Cdc2 was immunoprecipiated from the cell lysates using anti-Cdc2 (17) antibody and analyzed for Cdc2-Y15 phosphorylation and Cdc2 protein by immunoblotting.

Article Snippet: Recombinant p53 protein for ATM and ATR kinase assays was a glutathione S-transferase (GST) fusion protein containing full-length human p53 (Addgene, Cambridge, MA).

Techniques: Inhibition, Activation Assay, Protein-Protein interactions, Incubation, Immunoprecipitation, Activity Assay, Recombinant, Western Blot, Phospho-proteomics