Extended Figure 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.

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 tumor growth of 8p11^AMP and NSD3-overexpressing LUSC cell lines. Upper panel: Tumor volume quantification of human LUSC cell line xenografts described in (Figure 4a and Extended Data Fig 7a) treated with sgControl or sgNSD3 as indicated and grown in immunocompromised mice at 28 days after implantation (n = 5 mice, for each treatment group). P values indicated determined by one-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± s.e.m. Lower panel: Western blot analysis with the indicated antibodies of whole cell lysates from cells in upper panel. γH2AX levels are shown to assess whether sgNSD3 expression induces non-specific DNA damage. H3 and Actin are used as loading controls. (upper panel). c, NSD3 depletion by shRNA attenuates xenograft tumor growth of 8p11^AMP positive LUSC cell lines. Upper panel: Tumor volume quantification of the indicated 8p11^AMP-positive and two control 8p11^AMP-negative human LUSC cell line xenografts described 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 indicated determined by one-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± s.e.m. Lower panel: Western blot analysis with the indicated antibodies of whole cell lysates from cells in upper 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 (NSD3_TA) NSD3_{T1232A/Y1174A} (NSD3_TA/YA). Western blots of H520 lysates with indicated antibodies H3 and tubulin were used as loading controls (see Figure 4b). e, Western blots of whole cell lysate of AALE cells used in transformation assays for Figure 4c with ectopic expression of SOX2 and the indicated V5-tagged constructs (NSD3_WT, NSD3_TA, NSD3_YA, NSD3_TA/YA, NSD3_Short or FOXE1). f, Quantification of soft agar colony formation for AALE tracheobronchial epithelial cells with ectopic expression of SOX2 and NSD3_WT, NSD3_TA, NSD3_YA, NSD3_TA/YA, NSD3_Short 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 Figure 4c. Optical overlay of bioluminescent signal with X-ray image of mice grafted under the renal capsule with AALE cells expressing plasmids as in (e) and AkaLuc after substrate (AkaLumine-HCl; see Methods) administration (n = 5 for each condition). The color bar indicates the total bioluminescence radiance (photons/s/cm²/sr).