Fig 2: Opposing functions of LSD1 and SIRT1 in NHEJ repairA. Western blots of individual and combinatory knockdown of LSD1 and SIRT1. SCR, scrambled shRNA. B. NHEJ repair assay comparing individual and combinatory LSD1 and SIRT1 knockdown in KCL-22 cell-based NHEJ reporter cells. Bars represented mean percentage GFP positive cells with scrambled shRNA (shSP) or individual and combinatory gene knockdown. C. NHEJ reporter cells with SIRT1 or Scrambled (SCR) knockdown in the presence and absence of 50 or 200μM of the LSD1 inhibitor 2-PCPA. Dark gray bars represent percent of GFP cells without DOX induction, and light gray bars represent that with DOX induction. *P<0.01, **P<0.0001 and #P<0.01 when compared with SCR-DMSO; ♦P<0.01 compared with shSIRT1-DMSO. D. Left panel: immunoblots of NHEJ reporter cells transduced with vector alone, WT or K661A catalytically-inactive LSD1 expression vectors. HA-tagged LSD1 is detected using anti HA antibody. Right panel: comparing NHEJ repair with WT or K661A LSD1 over-expression. Bar graphs represented means (n=3) of percent GFP positive cells.

Fig 3: Lsd1 overexpression accelerates skeletal muscle regeneration. a Tibialis muscle mass normalized to body mass of control mice (Ctrl) or mice overexpressing LSD1 selectively in Myf5-positive muscle precursors (LSD1cTg) 5 days after Ctx or vehicle injection (left panel). Number of regenerating centronuclear fibers per area (mm2) in Ctrl and LSD1cTg mice 5 days after Ctx treatment (right panel). Significance was calculated by two-way ANOVA test. b Gomori staining of representative tibialis muscle sections from Ctrl and LSD1cTg mice 0, 5, and 7 days after Ctx treatment. c CSA measurement of regenerating centronuclear muscle fibers from Ctrl and LSD1cTg mice 5 and 7 days after Ctx treatment. Significance was calculated by two-way ANOVA test. d Immunofluorescence assay using antibodies directed against pan-myosin heavy chain (Myh, red) and green fluorescent protein (GFP, green) on control (Ctrl-GFP) and LSD1 overexpressing (LSD1-GFP) satellite cells isolated from extensor digitorum longus (EDL) fiber culture differentiated in myogenic medium for 0, 1, 3, or 7 days. Nuclei were stained with DAPI (blue). e Quantification of the nuclei per fiber shown in immunofluorescence assay in d. Significance was calculated by two-tailed Student’s t-test. (a, c: n = 5, e: n = 3; mean + SEM, NS = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001; scale bars: b, d 100 µm)

Fig 4: LSD1 inhibition reduces xCT expression via ATF4.A ATF4 mRNA levels were determined by qRT-PCR in NSCLC cell lines treated with 10 μM ORY for 96 h. B ATF4 expression was determined by western blotting in NSCLC cell lines treated with 10 μM ORY for 96 h. C The expression of xCT and ATF4 were measured by western blotting in ATF4-overexpressing A549 and H1975 cells treated with 10 μM ORY for 96 h. D ATF4 expression were measured by Western blotting in ATF4-overexpressing A549 and H1975 cells. E Cell viability was determined by SRB assay in ATF4-overexpressing A549 and H1975 cells treated with ORY at increasing concentrations for 96 h. F The expression of xCT and ATF4 were measured by western blotting in ATF4-overexpressing A549 and H1975 cells transfected with xCT siRNA. G, H After ATF4-overexpressing A549 and H1975 cells were transfected with xCT siRNA and were treated with ORY, cell viability (G) and the levels of GSH (H) were detected. I ChIP primers were designed spanning from −1000 to +400 bp around the transcription start sites of ATF4. J Quantitative ChIP studies were conducted to characterize the enrichment of H3K4me2 and H3K9me2 at promoters of ATF4 gene in A549 cells treated with 10 μM ORY for 96 h. n ≥ 3; Mean ± SEM; nsP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: LSD1-deficient epithelium renders mice susceptible to C. rodentium.(A) Survival plot of WT and cKO mice after C. rodentium infection. Kaplan-Meier survival curves was used and tested with a Gehan-Breslow-Wilcoxon test to identify significant differences between wild-type and cKO mice (p value = 0.0405). (B) Body weight loss comparing Naive, WT and cKO mice during C. rodentium infection. n = 9 (naive), n = 16 (WT) and n = 14 (cKO) mice, three pooled experiments. Data points are mean and SD. (C) Stool CFU (colony forming units) counts in WT (n = 22) and cKO (n = 21) infected mice using plating on agar after 6 days of C. rodentium infection. (D) CFU counts in liver and spleen tissue of WT (n = 12) and cKO (n = 11) mice at day 6 post C. rodentium infection. (E) RT-qPCR for Il22, Il17a and Ifng was carried out in colon tissue from naive (n = 10), WT (n = 12) and cKO (n = 11) mice infected with C. rodentium for 6 days. Expression is relative to Hprt. (F) ELISA for IL-22, IL-17A and IFN-γ in re-stimulated mesenteric lymph node cells after 6 days of C. rodentium infection. (G) Measurement of FITC dextran fluoroscence in serum derived from (naive = 4), WT (n = 4) and cKO (n = 4) infected with C. rodentium for 6 days. * P ≤ 0.05, ** P ≤ 0.01, Each dot represents one mouse, and means are depicted.