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. 2016 Oct 24;12(10):e1005916. doi: 10.1371/journal.ppat.1005916

Fig 5. Knockdown of Fascin does not influence cell aggregation between Jurkat T-cells and Raji/CD4+ B-cells.

Fig 5

(A-C) Conjugate formation between transfected Jurkat T-cells and co-cultured Raji/CD4+ B-cells was quantitated by flow cytometry. Jurkat T-cells were transfected with one of two different Tax-expression constructs (GFP-Tax; pEFTax) and one of two different shRNAs encoding IRES-EGFP and targeting Fascin (shFascin5, shFascin4) or a control (shNonsense). After 24h, Jurkat T-cells were co-cultured with Raji/CD4+ B-cells (ratio 1:1) for 1h at 37°C. Co-cultures were fixed and stained with anti-CD3-AlexaFluor700 (for Jurkat T-cells) and anti-HLA-DR-PacificBlue antibodies (for Raji/CD4+ B-cells) to differentiate between the two cell types. (A) Detection of GFP encoded by GFP-Tax and/or the shRNA construct (shNonsense) was used to differ between transfected (GFP+) and untransfected (GFP-) cells. Cell-cell conjugates were identified in GFP+ (Tax-positive) and GFP- (Tax-negative) gates as double-positive signals (HLA-DR+CD3+) and normalized on the total number of Jurkat T-cells (CD3+; see S6 Fig). The means of four independent experiments ± standard error (SE) are shown. Student’s t-tests were performed (*: p<0.05). (B) Control cells (shNonsense) compared to Fascin-repressed cells (shFascin5, shFascin4) within Tax-positive cells of A). (C) Detection of Fascin, GFP-Tax and Tax-1 in transfected Jurkat T-cells by western blot. β-actin (ACTB) served as control.