Fig 2: Kdm6b acts as a coactivator of Isl1-Lhx3.Kdm6b is associated with Lhx3 (a) or Isl1-Lhx3 (b) in co-immunoprecipitation assays in HEK293 cells. HEK293 cells were transfected with the constructs indicated on the left of the immunoblotting results. Antibodies used for immunoprecipitation were noted on the top. HA or Flag antibodies were used for immunoblotting assays, as shown on the right. The assays were performed three times in each condition and the representative results were shown. c Luciferase reporter assays in HEK293 cells transfected with HxRE:LUC or control LUC vector with (+) or without (-) Isl1-Lhx3, Kdm6b or Kdm6a, as indicated on the graph. Kdm6b enhanced Isl1-Lhx3-mediated transcriptional activity in a dose-dependent manner. n = 3 biologically independent samples. Error bars represent the standard deviation of the mean. d ChIP assays in E12.5 spinal cords with IgG or Kdm6b antibody. Kdm6b was recruited to the Isl1-Lhx3 binding loci in Mnx1, Isl1, and Lhx4 genes. The Untr6 was used as a negative control region. n = 3 biologically independent samples. Error bars represent the standard deviation of the mean. e The model depicts Kdm6b action in the development of spinal MNs. Kdm6b is recruited to the Isl1-Lhx3-binding hexamer response element (HxRE) by associating with Isl1-Lhx3 complex and activates the Isl1-Lhx3 target genes, including Isl1/2, Lhx3/4, and Mnx1 genes. This transcriptional activation promotes nbMN and MMC fates, in part by further enhancing Isl1-Lhx3 activity. Moreover, as Lhx3 inhibits Foxp1 expression, Kdm6b-mediated transcriptional activation indirectly suppresses LMC and PGC fates. Thus, Kdm6b controls the balanced production of MN subtypes.

Fig 3: Inhibition of JMJD3 improved acute lung injury by down-regulating ADORA2A thereby promoting apoptosis of macrophages.A The relationship between ADORA2A and cleaved caspase-3 in western blots after Adora2a knockdown in LPS-stimulated RAW264.7 cells: ADORA2A expression in the LPS group was higher than that in both the control group and GSK-J4 group. Compared to cells that were only exposed to LPS, cells that were further treated with GSK-J4, underwent knockdown of Adora2a, or were both treated with GSK-J4 and underwent Adora2a knockdown showed higher levels of cleaved caspase 3 (n = 3 in each group, ADORA2A: P < 0.001, control vs. LPS group, P < 0.001, LPS+GSK-J4 vs. LPS group, P < 0.001, LPS+Adora2a knockdown vs. LPS group, P < 0.001, LPS+Adora2a knockdown+GSK-J4 vs LPS group. Cleaved caspase-3: P < 0.05, control vs. LPS group, P < 0.01, LPS+GSK-J4 vs. LPS group, P < 0.01, LPS+Adora2a knockdown vs. LPS group, P < 0.01, LPS+Adora2a knockdown+GSK-J4 vs. LPS group). B Flow cytometry analysis for the evaluation of apoptosis after Adora2a knockdown in LPS-stimulated RAW264.7 cells: Compared to cells that were only exposed to LPS, cells that were further treated with GSK-J4, underwent knockdown of Adora2a, or were both treated with GSK-J4 and underwent Adora2a knockdown showed higher levels of apoptosis (n = 3 in each group, P < 0.01, LPS+GSK-J4 vs. LPS group, P < 0.01, LPS+Adora2a knockdown vs. LPS group, P < 0.01, LPS+Adora2a knockdown+GSK-J4 vs. LPS group). ns indicated no statistical significance, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 4: JMJD3 was upregulated in lung tissue of ALI and LPS-stimulated macrophage.A JMJD3 expression level in the LPS group was higher than that in the control group but lower than that in the GSK-J4 treatment group (n = 10 in each group, P < 0.01, control vs. LPS group, P < 0.01, LPS vs. LPS+GSK-J4 group). B Western blot shows that in macrophage, the JMJD3 expression level in the LPS group was higher than that in the control group (P < 0.01, control vs. LPS group), using GSK-J4 could inhibit JMJD3 in macrophage (n = 10 in each group, P < 0.01, LPS vs. LPS+GSK-J4 group). C Transcription level of Jmjd3 in the LPS group was higher than that in the control group (P < 0.01, control vs. LPS group) but lower than that in GSK-J4 treatment group (P < 0.01, LPS vs. LPS + GSK-J4 group). ns indicated no statistical significance, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: KDM6A protein levels are decreased following EMT and upregulated during MET(A) HMLE cells were transduced with either an empty vector (HMLE-Vector), or genes encoding the EMT-inducing transcription factors Snail (HMLE-Snail) or Twist (HMLE-Twist) [4]. Whole-cell lysates were analyzed by immunoblotting for KDM6A. ß-actin was used as a loading control. (B-F) MCF10A cells were treated with sterile 4 mM HCl containing 0.1% bovine serum albumin (Vehicle) or TGFB for 5 days (TGFB+). In parallel, MCF10A cells were exposed to TGFB for 5 days in order to induce EMT, and subsequently subjected to TGFB-withdrawal, for a further 5 days, to elicit MET (TGFB+/-). (B-C) MCF10A cells, treated as described, were fixed and immunostained for E-cadherin (B) and N-cadherin (C). Nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. (D) MCF10A cells, treated as described, were analyzed by immunoblotting for KDM6B and vimentin (Vim). COX IV was used as a loading control. (E) MCF10A cells, treated as described, were fixed and immunostained for KDM6A. Nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. (F) Cells were analyzed by immunoblotting for KDM6A, EZH2 and RING1A. ß-actin was used as a loading control.