Fig 1: Knockdown of KDM1A suppresses osteogenic differentiation of BM-MSCs (A) BM-MSCs were transfected with shKDM1A_1–4# or shNC and the expression level of KDM1A in shKDM1A_1–4# or shNC transfected BM-MSCs was detected using western blot. BM-MSCs that were transfected with shKDM1A or shNC were cultured in OM or NM for 14 days. Afterward, different assays were performed to assess the osteogenic differentiation. (B) Representative images of ARS and ALP staining. Scar bar = 100 µm. (C) Representative RUNX2 immunofluorescence staining image. Scar bar = 100 µm. Gene and protein expression of osteogenic markerslivergent, ALP, OPN, RUNX2 and Colla1, and circ_AFF4, FNDC5 were detected by (D) RT-qPCR and (E) western blot. (F) ELISA detected Irisin expression. *p < 0.05, **p < 0.001 and ***p < 0.001. Each experiment was performed at least three times independently.
Fig 2: OGD/R induces pyroptosis of RGCs and upregulates LSD1. RGC-5 cells received OGD/R treatment. A: RGC-5 cell viability was detected via the CCK-8 method; B: The concentrations of IL-1β and IL-18 were examined via ELISA; C: The mRNA levels of LSD1 and Caspase 1 in RGC-5 cells were determined via RT-qPCR; D: The protein levels of NLRP3, GSDMD-N, cleaved-Caspase1, and LSD1 in RGC-5 cells were examined via Western blot analysis. Each cell experiment was performed in triplicate; measurement data were presented as mean ± standard deviation (SD); data in panel A were analyzed via the t-test; data in panels B-D were analyzed via two-way ANOVA, followed by Tukey’s multiple comparison test; *p < 0.05
Fig 3: High levels of LSD1 in cone photoreceptors but not rods. Immunofluorescence staining of P36 C57BL/6J mouse retinas for LSD1 (green), short wavelength cone opsin (red), and DAPI nuclear stain (blue). The 40× merged image taken with a confocal microscope showed LSD1 expression in all three nuclear layers and short wavelength cone opsin expression in cone photoreceptor outer segments (A). The 60× image shows perfect correlation between cells with high LSD1 expression (green) along the outer edge of the ONL and the cone opsin (red) (B).
Fig 4: LSD1 substrates H3K4me1 and H3K4me2 are expressed throughout the retinoblast and mature retina. Immunofluorescence staining of C57BL/6J mouse retinas at P2 and P36 for H3K4me1 and H3K4me2 (green) with a DAPI nuclear stain (blue). Images were taken using a 40× objective lens on a confocal microscope. At P2, H3K4me1 (A, B) and H3K4me2 (C, D) were expressed uniformly in the retinoblast throughout all RPCs. At P36 in the mature retina H3K4me1 (E, F) and H3K4me2 (G, H) maintain uniform expression in all retinal cell subtypes. GCL, ganglion cell layer; NBL, neuroblastic layer; RPE, retinal pigment epithelial layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Fig 5: LSD1 substrates H3K4me1 and H3K4me2 peak at P2 and significantly decrease across retinal developmental time. Western blot analysis was conducted on C57BL/6J mouse retina samples at five different time points (P2, P7, P14, P21, and P36) in triplicate. Samples were probed with an anti-H3K4me1 (A) or anti-H3K4me2 antibody (E) (single band, expected size, 18 kDa), and an anti-H3 antibody (B, F) (single band, expected size, 18 kDa) served as a loading control. Quantification of results was achieved using densitometry, and H3K4me1/H3K4me2 levels were normalized to H3. A two-way ANOVA with Tukey's multiple comparison test was conducted between the mean expression level in all possible pair combinations. For H3K4me1, there is a statistically significant decrease between P2 and P14, P2 and P21, P7 and P14, and P7 and P21 (D). For H3K4me2, there is a statistically significant decrease between P2 and P14, P2 and P21, P2 and P36, P7 and P14, P7 and P21, and P7 and P36 (H). A full list of comparisons and P values are listed in Supplementary Table S2 and Supplementary Table S3. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade