non target control shrna shctr  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Chromosome 8 gain drives poor patient outcome via expression of 4E-BP1 in Ewing sarcoma

doi: 10.1101/2022.12.11.519935

Figure Lengend Snippet: a Areaproportional Venn diagram of gene sets enriched with EIF4EBP1 expression in cohort 1 (A) and 2 (B) as well as with chr8 gain in cohort 2 (C) as determined by fGSEA. Exemplary gene sets representing a proliferation-associated enrichment signature in the overlap between A, B, and C were shown with respective normalized enrichment scores (NES) and significance levels. b fGSEA enrichment plots of exemplary gene sets given in (a). c Relative viable cell count of A-673, SK-N-MC, and TC-71 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr). Cells were grown either with or without Dox for 96h. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 4 biologically independent experiments. d Sphere formation in A-673, SK-N-MC, and TC-71 cells containing shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr) treated with or without Dox for 8–14 d. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 3 biologically independent experiments. Two-tailed unpaired t-test with Welch’s correction. Representative images of spheres are shown on the right. e Relative colony number of A-673 and SK-N-MC cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a nontargeting shControl (shCtr). Cells were grown either with or without Dox for 8–14 d. Horizontal bars represent means, and whiskers the SEM, n ≥ 4 biologically independent experiments. Representative images of colony formation are shown on the right. f Kaplan-Meier analysis of event-free survival of NSG mice xenografted with A-673 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr). Once tumors were palpable, mice were randomized and treated with either vehicle (–) or Dox (+), n ≥ 5 animals per condition. An ‘event’ is recorded when tumors reached a size maximum of 15 mm in one dimension. P -values determined via Mantel-Haenszel test. g Quantification of mitoses in HE-stained slides of xenografts described in (f). Five high-power fields (HPF) were counted per sample. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 4 samples per condition. *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant; P -values determined via two-tailed Mann-Whitney test if not otherwise specified.

Article Snippet: Human EwS cell lines A-673, SK-N-MC, and TC-71 were transduced with lentiviral Tet-pLKO-puro all-in-one vector system (plasmid #21915, Addgene) containing a puromycin-resistance cassette, and a tet-responsive element for Dox-inducible expression of shRNAs against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting control shRNA (shCtr).

Techniques: Expressing, Cell Counting, shRNA, Construct, Two Tailed Test, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chromosome 8 gain drives poor patient outcome via expression of 4E-BP1 in Ewing sarcoma

doi: 10.1101/2022.12.11.519935

Figure Lengend Snippet: a Relative EIF4EBP1 expression in 21 wild-type EwS cell lines as determined by qRT-PCR. b Relative EIF4EBP1 expression as assessed by qRT-PCR in A-673, SK-N-MC, and TC-71 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr). Cells were grown either with or without Dox for 96h. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 3 biologically independent experiments. c Relative 4E-BP1 expression as assessed by quantified western blotting in A-673, SK-N-MC, and TC-71 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr). Cells were grown either with or without Dox for 96h. P -values determined via one-tailed Mantel-Haenszel test. d Representative western blots as described in (b) are shown. ß-actin served as a loading control. e Relative number of dead cells as assessed by Trypan blue exclusion in A-673, SK-N-MC, and TC-71 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting shControl (shCtr). Cells were grown either with or without Dox for 96h. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 4 biologically independent experiments. f Kaplan-Meier analysis of event-free survival of NSG mice xenografted with TC-71 cells containing Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2). Once tumors were palpable, mice were randomized and treated with either vehicle (–) or Dox (+), n =8 animals per condition. An ‘event’ is recorded when tumors reached a size maximum of 15 mm in one dimension. P -values determined via Mantel-Haenszel test. g Quantification of mitoses in HE-stained slides of xenografts described in (f). Five high-power fields (HPF) were counted per sample. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 7 samples per condition. h Quantification of necrotic area on HE-stained slides of A-673 xenografts described in (f). Five high-power fields (HPF) were analyzed per sample. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 5 samples per condition. i Quantification of necrotic area on HE-stained slides of TC-71 xenografts described in (f). Five high-power fields (HPF) were analyzed per sample. Horizontal bars represent means, and whiskers represent the SEM, n ≥ 7 samples per condition. j Kaplan-Meier analysis of event-free survival of NSG mice orthotopically xenografted into the proximal tibia with TC-71 containing a Dox-inducible specific shRNA construct directed against EIF4EBP1 (sh4E-BP1_2). One day after injection of the cells, mice were randomized and treated with either vehicle (−) or Dox (+), n =5 animals per condition. An ‘event’ is recorded when the mice exhibited signs of limping at the injected leg. P -values determined via Mantel-Haenszel test. *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant; P values determined via two-tailed Mann-Whitney test if not otherwise specified.

Article Snippet: Human EwS cell lines A-673, SK-N-MC, and TC-71 were transduced with lentiviral Tet-pLKO-puro all-in-one vector system (plasmid #21915, Addgene) containing a puromycin-resistance cassette, and a tet-responsive element for Dox-inducible expression of shRNAs against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or a non-targeting control shRNA (shCtr).

Techniques: Expressing, Quantitative RT-PCR, shRNA, Construct, Western Blot, One-tailed Test, Staining, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Experimental Hematology & Oncology

Article Title: Targeting phosphoglycerate dehydrogenase in multiple myeloma

doi: 10.1186/s40164-020-00196-w

Figure Lengend Snippet: PHGDH knockdown potentiates bortezomib and proteasome-resistant cell lines are sensitive to PHGDH inhibition. a The knockdown of PHGDH using shRNA was confirmed via WB. b CTG was used to highlight the dependence of the shPHGDH cells on extracellular serine. The sensitivity to drugs was tested by CTG. c , d INA6 KD cells were treated overnight with carfilzomib and bortezomib, respectively. e , f INA6-res and AMO1-res cell lines, respectively, were treated with the indicated doses of NCT-503 overnight. All the presented graphs and calculated IC50s represent three independent experiments with minimum two replicates. Error bars are ± SEM and * P ≤ 0.05 , ** P ≤ 0.01, *** P ≤ 0.001

Article Snippet: Following the manufacturer’s protocol, INA6 knockdown cells (INA6-KD) were transduced with lentiviral particles containing either non-target control shRNA (shCTR) or shRNA targeting PHGDH (shPHGDH) (Santa Cruz Biotechnology, Dallas, TX, USA; sc-108080 and sc-105011-V).

Techniques: Knockdown, Inhibition, shRNA