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: Nature communications

Article Title: Global translation during early development depends on the essential transcription factor PRDM10.

doi: 10.1038/s41467-020-17304-3

Figure Lengend Snippet: Fig. 4 Loss of EIF3B causes lethality in preimplantation embryos and mESCs. a Eif3b-deficient embryos arrest prior to blastocyst formation. Expression of candidate target genes was knocked down in wild-type zygotes by injection of siRNA: control (n = 38), Eif3b (n = 40), Selenow (n = 93), Ss18 (n = 87), and Ube2a (n = 66), and blastocyst morphology was scored after 4 days’ embryo culture. Y-axis: percentage of cavitated or non-cavitated blastocysts of total embryos analyzed in each experiment, for at least 3 independent experiments per target gene. b Representative images of embryos treated with control or Eif3b-targeting siRNA, acquired at 4.5 dpc. Scale bar: 100 μm. c Western blot analysis of E14 mESCs overexpressing Eif3b or vector control, transduced with indicated shRNA. Numbers in bottom row represent quantification of relative EIF3B protein levels after background subtraction and normalization to α- tubulin. d qRT-PCR validation of Eif3b knockdown in E14 mESCs transformed with vector control (vector) or Eif3b overexpression construct (Eif3b OE) and transduced with shRNA targeting the 3′UTR of Eif3b (shRNA-479). 3′UTR-specific primers were used to detect endogenous Eif3b transcript. n = 2 samples, data shown from one out of three independent experiments with similar results. e E14 mESCs transduced with shRNA-479 to deplete endogenous Eif3b exhibit slower growth (vector + 479; black dashed line), while expression of shRNA-resistant Eif3b restores normal growth (Eif3b OE + 479; red dashed line). Cells were plated at equal densities, passaged at Day 2 and Day 4, and counted at indicated time-points. n = 3 replicates, representative data shown (from same experiment as in Fig. 4d). Error bars denote mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant; two-tailed unpaired Student’s t test (a and d), two-way ANOVA with Tukey’s multiple comparisons test (e).

Article Snippet: For the construction of shRNA lentiviral vectors, gene-specific hairpin sequences were selected from the RNAi Consortium TRC lentiviral shRNA library and cloned into pLKO.1-Neo (Addgene, #13425) as annealed oligonucleotides (Supplementary Data 8).

Techniques: Expressing, Injection, Control, Embryo Culture, Western Blot, Plasmid Preparation, Transduction, shRNA, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Transformation Assay, Over Expression, Construct, Two Tailed Test