Fig 2: NSD1/2 and LEDGF/HDGF2 are required for H3K27M-DIPG cell proliferation.(A) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. (B) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. (C) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). (D) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. (E) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. (F) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Fig 3: NSD3 is essential for H3K36 methylation, oncogenic genes expression and mTOR activation in pancreatic cancer cells.Stable PanCa-1 cells, bearing shNSD3-S5, the scramble control shRNA (“shC”), the lenti-CRISPR/Cas9 construct encoding NSD3 sgRNA (“koNSD3”) or the lenti-CRISPR/Cas9 empty vector (“koC”), were established and cultured; expression of listed proteins was tested by western blotting assays (A, C); listed mRNAs were examined by qRT-PCR assays (B). Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “shC” cells. Experiments in this figure were repeated five times with similar results obtained.

Fig 4: NSD3 shRNA injection inhibits PanCa-1 xenograft tumor growth in nude mice.The PanCa-1 xenograft-bearing nude mice were subjected to intratumoral injection of AAV-packed shNSD3-S4 (“aav-shNSD3-S4”), AAV-packed-shNSD3-S5 (“aav-shNSD3-S5”) or AAV-packed shC (“aav-shC”), daily for 7 consecutive days; tumor volumes (A) and mice body weights (D) were recorded every 7 days for a total of 42 days. The estimated daily tumor growth, in mm3 per day, was calculated using the described formula (B). All tumors were carefully isolated at Day-42 and weighted individually (C). At Day-7 and Day-14, one tumor of each group was carefully separated and total six tumors were acquired. Expression of listed mRNAs and proteins in tumor tissue lysates were tested by qRT-PCR (E, G) and western blotting (F, H) assays, respectively. Targeted proteins were quantified and normalized to the corresponding loading controls (F, H). Data were presented as mean ± standard deviation (SD). N = 12 stands for 12 mice per group (A–D). *P < 0.05 vs. “aav-shC” group.

Fig 5: NSD3-T1232A accelerates pancreatic cancer cell proliferation, migration and invasion.Single stable PanCa-1 primary pancreatic cancer cells bearing the lenti-CRISPR/Cas9 construct encoding NSD3 sgRNA (“koNSD3”) were further transduced with NSD3-T1232A-Flag construct, the wild-type NSD3-Flag construct or empty vector (“Vec”); Expression of listed proteins was examined by western blotting assays (A). Cells were further cultured for applied time periods, cell viability (CCK-8 assays, B), proliferation (EdU staining assays, C), cell migration (“Transwell” assays, D), and invasion (“Matrigel Transwell” assays, E) were tested. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05. Experiments in this figure were repeated five times with similar results obtained. Scale bar = 100 μm (C–E).