Validation of LINC perturbation on NCT using CRISPR interference. (A–C) IF validation of transient loss of LINC proteins in BJ-5ta cells stably expressing dCas9-KRAB-IRES-LINuS transfected with guide RNAs against (A) Nesprin1 (sgNesprin1), (B) Nesprin2 (sgNesprin2), or (C) Vimentin (sgVimentin). Scale bars 10 µm. (D) Western blot validation of the transient loss of SUN1 (sgSUN1) or SUN2 (sgSUN2) proteins in BJ-5ta cells stably expressing dCas9-KRAB-IRES-LINuS. Tubulin was used as a loading control. (E and F) Import and export rates of BJ-5ta cells expressing KRAB-dCas9-IRES-LINuS (CRISPRi) and transfected with gRNAs against SUN1 (sgSUN1), SUN2 (sgSUN2), Nesprin1 (sgNesprin1), Nesprin2 (sgNesprin2), and vimentin (sgVimentin). sgControl (n = 109), sgSUN1 (n = 106), sgSUN2 (n = 80); three independent replicates. sgVimentin (n = 42), sgNesprin1 (n = 25), sgNesprin2 (n = 24); two independent replicates. Significance was calculated using one-way ANOVA with Dunnett’s post hoc. ns P > 0.05, P***<0.001, P****<0.0001. Source data are available for this figure: SourceData FS3.