rnai consortium (trc) shrna library  (Broad Institute Inc)

Journal: bioRxiv

Article Title: Modelling oxaliplatin resistance in colorectal cancer reveals a SERPINE1 -based gene signature (RESIST-M) and therapeutic strategies for pro-metastatic CMS4 subtype

doi: 10.1101/2024.12.17.628817

Figure Lengend Snippet: Immunoblotting of PAI-1 protein in HCT116 MD and HD knocked down for the protein using two unique shRNAs ( A ). Fold change of PAI-1 protein in cell culture supernatant of HCT116 MD and HD knocked down for the protein relative to MD shRNA control (shCtrl) ( B ). Fold change of dose response to 48 hrs oxaliplatin treatment in HCT116 MD and HD knocked down for the protein relative to MD shCtrl ( C ). Representative dose response curves of HCT116-MD shCtrl, PAI-1 sh3 and sh5 ( D ) and HCT116-HD shCtrl, PAI-1 sh3 and sh5 ( E ) to 48 hrs of oxaliplatin treatment. F-F’, Inhibition of metastasis in oxaliplatin treated HCT116 model using tiplaxtinin. Three representative H&E staining and corresponding quantifications ( F and F’ ) of lung sections from mice harboring HCT116 PAR xenografts treated with oxaliplatin, alone or in combination with tiplaxtinin, until spontaneous metastasis (refer to section ‘Impact of concomitant administration of tiplaxtinin and oxaliplatin on metastasis’ under Materials and Methods). For quantification number of nodule positive area was normalized to total lung area (n= at least 8). Statistical significance was determined using Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test for Fig. B, C, and using two-tailed unpaired t-test for Fig. F’. A p-value of <0.05 was considered significant for all analyses, unless stated otherwise.

Article Snippet: To generate stable knockdown lines, suitable bacteria clones from the RNAi Consortium (TRC) shRNA Library (Broad Institute) were selected and sub-cultured in terrific Broth (CUS-4051-1L, Axil Scientific) supplemented with 1X carbenicillin (10177012, ThermoFisher).

Techniques: Western Blot, Cell Culture, shRNA, Control, Inhibition, Staining, Two Tailed Test

Journal: PLoS ONE

Article Title: SHP-1 Phosphatase Is a Critical Regulator in Preventing Natural Killer Cell Self-Killing

doi: 10.1371/journal.pone.0044244

Figure Lengend Snippet: A. Efficient SHP-1 gene silencing in primary murine IL-2-activated NK cells. Purified C57BL/6NCrl IL-2 activated NK cells were transduced on two consecutive days with TRC lentiviral vectors and incubated for 3 days post-transduction. Transduced cells were puromycin selected for 48 hours followed by 3 days incubation. Cells were assayed for SHP-1 expression by intracellular staining with primary rabbit anti-SHP-1 and secondary anti-rabbit Alexa Fluor 488 antibodies in flow cytometry (Black). Secondary antibodies alone (Grey) was used as negative control. Data is representative of 3 experiments. B. IL-2 activated NK cells were transduced on two consecutive days with mock, shEGFP and SHP-1 shRNA and incubated for 3 more days. Transduced cells were puromycin-selected and used as effectors against Yac-1, P815 and RMA-S targets at E:T ratio 1∶1 in CD107a cytotoxicity assay. Background degranulation responses of the NK effectors (in the absence of target cells) were subtracted from the degranulation responses observed in the presence of the target cells. Data of three independent experiments were pooled together for statistical analyses. N.S., (p>0.05), non significant.

Article Snippet: We searched the RNAi Consortium (TRC) lentiviral shRNA library (Open Biosystems, Thermo Fisher Scientific) for potential shRNA sequences targeting against the mouse SHP-1 gene (TRCN0000028964 - 68).

Techniques: Purification, Incubation, Transduction, Expressing, Staining, Flow Cytometry, Negative Control, shRNA, Cytotoxicity Assay