Extended Data Fig. 7: NSD3 depletion attenuates growth of 8p11-amplified and NSD3-overexpressing human LUSC cell lines and ectopic expression of NSD3 cooperates with SOX2 to transform tracheobronchial epithelial cells.
From: Elevated NSD3 histone methylation activity drives squamous cell lung cancer
a, Genetic alterations and mRNA expression of LUSC-associated genes in human LUSC cell lines used in this study. b, NSD3 depletion by CRISPR–Cas9 attenuates xenograft tumour growth of 8p11AMP and NSD3-overexpressing LUSC cell lines. Top, tumour volume quantification of human LUSC cell line xenografts described in Fig. 4a and Extended Data Fig. 7a treated with sgControl or sgNSD3 and grown in immunocompromised mice at 28 days after implantation (n = 5 mice for each treatment group). P values determined by one-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± s.e.m. Bottom, western blot analysis with the indicated antibodies of whole-cell lysates from cells in top panel. γH2AX levels are shown to assess whether sgNSD3 expression induces non-specific DNA damage. H3 and actin are used as loading controls. c, NSD3 depletion by shRNA attenuates xenograft tumour growth of 8p11AMP-positive LUSC cell lines. Top, tumour volume quantification of the indicated 8p11AMP-positive and two control 8p11AMP-negative human LUSC cell line xenografts as in a treated with shControl or shNSD3 as indicated and grown in immunocompromised mice at 28 days after implantation (n = 5 mice for each treatment group). P values determined by one-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± s.e.m. Bottom, western blot analysis with the indicated antibodies of whole-cell lysates from cells in top panel. Actin is used as a loading control. d, NSD3-deficient H520 cells reconstituted with the indicated V5-tagged CRISPR-resistant NSD3 derivatives: NSD3(T1232A) (NSD3TA) and NSD3(T1232A/Y1174A) (NSD3TA/YA). Western blots of H520 lysates with indicated antibodies H3 and tubulin were used as loading controls (Fig. 4b). e, Western blots of whole-cell lysate of AALE cells used in transformation assays for Fig. 4c with ectopic expression of SOX2 and the indicated V5-tagged constructs (NSD3WT, NSD3TA, NSD3YA, NSD3TA/YA, NSD3Short or FOXE1). f, Quantification of soft agar colony formation for AALE tracheobronchial epithelial cells with ectopic expression of SOX2 and NSD3WT, NSD3TA, NSD3YA, NSD3TA/YA, NSD3Short or FOXE1 as in e. Data are represented as mean ± s.e.m. of three technical replicates in two independent experiments. g, Representative soft-agar images from AALE transformation assays in f. h, In vivo AALE transformation assay images from Fig. 4c. Optical overlay of bioluminescent signal with X-ray images of mice grafted under the renal capsule with AALE cells expressing plasmids as in e and AkaLuc after substrate (AkaLumine-HCl; Methods) administration (n = 5 for each condition). The colour bar indicates the total bioluminescence radiance (photons/s/cm2/sr).
