control plasmid sh-scramble  (VectorBuilder GmbH)

Journal: Molecular Biology of the Cell

Article Title: The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly

doi: 10.1091/mbc.e23-02-0065

Figure Lengend Snippet: Figure 8. Loss of EBP50 results in severe BB assembly defects in CACO-2BBE cells. (A) Confocal images of 12-day polarized CACO-2BBE cells stably expressing either a scramble shRNA construct or shRNAs targeting EBP50 (KD 37 and 64), stained for EBP50 (green) and F-

Article Snippet: Knockdown studies utilized a non-targeting scramble control shRNA (Addgene; plasmid #46896), CDHR5 KD shRNA (TRC clone TRCN000054168; KD68), and EBP50 KD shRNA clones (TRCN0000043733; KD33), (TRCN0000043734; KD34), (TRCN0000043735; KD35), (TRCN0000043737; KD37), (TRCN0000440444; KD44) and (TRCN0000437164; KD64) sequences that were expressed from the pLKO.1 plasmid.

Techniques: Stable Transfection, Expressing, shRNA, Construct, Staining

Journal: Molecular Biology of the Cell

Article Title: The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly

doi: 10.1091/mbc.e23-02-0065

Figure Lengend Snippet: Figure 9. Loss of CDHR5 results in BB assembly defects in CACO-2BBE cells. (A) Confocal images of 12-day polarized CACO-2BBE cells stably expressing either a scramble shRNA construct or an shRNA targeting CDHR5, stained for CDHR5, EBP50, Ezrin, and Phospho-ERM (P-ERM) (green) and F-actin (red). Boxed regions denote the area in zoomed image panels. Scale bars, 10 μm. Loss of CDHR5 expression disrupts proper apical assembly, but does not block apical targeting of Ezrin, activated Ezrin (detected by P-ERM antibody) and EBP50. (B) Scatterplot quantification of apical/total signal ratios of Ezrin, activated Ezrin (P-ERM) and EBP50 in scramble and CDHR5 KD CACO-2BBE cells. Each data point represents the ratio of the entire apical signal found in the x-z section compared to the total signal of the x-z section. Measurements of apical to total signal: EBP50 signal, scramble n=78, KD n=65; Ezrin signal, scramble n=74, KD n=56; P-ERM signal, scramble n=62, KD n=69. ns = not significant, *p < 0.01, two-tailed t test. (C) Cartoon proposing the new interactome of the IMAC, in which CDHR5 is cross-linked to the actin cytoskeleton via EBP50-Ezrin.

Article Snippet: Knockdown studies utilized a non-targeting scramble control shRNA (Addgene; plasmid #46896), CDHR5 KD shRNA (TRC clone TRCN000054168; KD68), and EBP50 KD shRNA clones (TRCN0000043733; KD33), (TRCN0000043734; KD34), (TRCN0000043735; KD35), (TRCN0000043737; KD37), (TRCN0000440444; KD44) and (TRCN0000437164; KD64) sequences that were expressed from the pLKO.1 plasmid.

Techniques: Stable Transfection, Expressing, shRNA, Construct, Staining, Blocking Assay, Two Tailed Test

Journal: International Journal of Genomics

Article Title: Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 3 Expression and Its Correlation with Prognosis and Growth of Serous Ovarian Cancer: Correlation of DYRK3 with Ovarian Cancer Survival

doi: 10.1155/2024/6683202

Figure Lengend Snippet: Cellular effects of DYRK3 knockdown in ovarian cancer cells. (a, b) MTT assay showing decreased cell growth in both CAOV3 and OVCAR-3 cell lines following DYRK3 knockdown by shRNAs. Control cells treated with scrambled-shRNA or mock control exhibit higher growth rates. (c, d) Matrigel-Transwell assay demonstrating impaired invasion capacities in both CAOV3 and OVCAR-3 cell lines upon DYRK3 knockdown. ∗ indicates P < 0.05.

Article Snippet: For transfection, shRNAs targeting DYRK3 or scramble control shRNA were utilized, following the manufacturer's instructions (Cat. sc-39010-SH; Santa Cruz Biotechnology).

Techniques: Knockdown, MTT Assay, Control, shRNA, Transwell Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion

doi: 10.3389/fcell.2022.898088

Figure Lengend Snippet: Decreased ISG15 expression alters HTR8/SVneo cell morphology and reduces F- actin levels. (A–D) ISG15 mRNA (A) and protein levels (B) as well as cell morphology (C) and TRITC-conjugated Phalloidin (red) fluorescence stained F-actin levels (D) are represented in ISG15 -siRNA vs. control- siRNA transfected HTR8/SVneo cells. Bars represent Mean ± SEM and compared by using t -test, n = 3, Scale bars = 30 µm (C) , Scale bars = 6 µm (D).

Article Snippet: The following day, cells were transiently transfected with either ISG15 -specific or non-specific scrambled control siRNA (Santa Cruz) in Opti-MEM media (Invitrogen) using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Staining, Control, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion

doi: 10.3389/fcell.2022.898088

Figure Lengend Snippet: ISG15 silencing reduces trophoblast migration, invasion and proliferation capacities. (A) In vitro wound healing (0–44 h), (B) migration ( n = 5, 48 h), and (C) invasion ( n = 5, 60 h) capacities, mRNA levels of (D) ITGB1 ( n = 3), (E) ITGB4 genes ( n = 3), as well as (F) proliferation ( n = 5, 48 h) and (G) apoptotic index ( n = 6, 48 h) are compared in control- vs. ISG15 -siRNA transfected HTR8/SVneo cells. Bars represent Mean ± SEM and compared by using t -test. Scale bar = 500 µm.

Article Snippet: The following day, cells were transiently transfected with either ISG15 -specific or non-specific scrambled control siRNA (Santa Cruz) in Opti-MEM media (Invitrogen) using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Migration, In Vitro, Control, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion

doi: 10.3389/fcell.2022.898088

Figure Lengend Snippet: ISG15 silencing amplifies IL-1β induced expression of pro-inflammatory cytokines. (A–D) Expression levels of IL1B , CXCL8 , IL6 , and CCL2 mRNA in control vs. ISG15 -siRNA transfected HTR8/SVneo cells, followed by 6 h treatment with vehicle (vh) or 10 ng/ml IL1β (IL-1β). (E,F) IL-6 and MCP-1, aka CCL2 , protein secretion levels in ISG15 -siRNA vs. control transfected HTR8/SVneo cells treated with 10 ng/ml IL1β (IL-1β). (A–D) : Bars represent median values and compared by using One way ANOVA, n = 4, * p = 0.007, Φ P = 0.005, ¥ p = 0.008, # p = 0.003. (E,F) : Bars represent Mean ± SEM and compared by using t -test ( n = 4).

Article Snippet: The following day, cells were transiently transfected with either ISG15 -specific or non-specific scrambled control siRNA (Santa Cruz) in Opti-MEM media (Invitrogen) using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Transfection