Fig. 5
Loss of mCa2+ uptake drives myofibroblast differentiation through epigenetic reprogramming. a Simplified schematic of the reaction mechanism of α-ketoglutarate (αKG)-dependent dioxygenases: ten-eleven translocation (TET) enzymes and Jumonji-C (JmjC)-domain-containing demethylases (JmjC-KDMs). b Levels of 5-methylcytosine (5-mC) were measured in Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs by ELISA. Fold change vs. Ad-βgal veh. c MEFs were treated with TGFβ for 0, 12 or 24 h and cell lysates were immunoblotted for specific methylated histone 3 lysine (H3K) residues. Total H3 and Tubulin were used as loading controls. d Quantification of H3K27me2 protein expression. Band density was normalized to total H3. e H3K27me2 chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) of Periostin in Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs at baseline (veh) and following 12 h TGFβ. Schematic shows loci of qPCR primers in relationship to myofibroblast-associated transcription factor binding sites – NFAT (nuclear factor of activated T-cells), SRF (serum response factor). f Expression of periostin (Postn) mRNA in Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs at baseline (veh) and post-TGFβ. g WT MEFs were treated with vehicle or TGFβ and assessed for chromatin accessibility and transcription using ATAC-seq and RNA-seq. Results of the Postn locus are shown. The height of the genome browser tracks shows the number of reads normalized by read depth and overall peak enrichment in the library. h–j Wildtype MEFs treated +/− cell-permeable, dimethyl-αKG and +/− TGFβ for 48 h followed by immunofluorescence for α-SMA. Representative images and quantification of percentage of α-SMA+ cells are shown. k Schematic of JmjC-KDM reactions indicating the specific JmjC-KDMs inhibited by JIB-04. l–o Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs were treated with vehicle, TGFβ, or TGFβ + 1 μM JIB-04 for 24 h and immunofluorescence was performed by costaining with α-smooth muscle actin (α-SMA) antibody (red) and DAPI (blue). Representative images and quantification of percentage of α-SMA+ cells are shown. n = 3 experiments for all quantified data. All data shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle control analyzed by ANOVA. ###p < 0.001, ##p < 0.01, #p < 0.05 vs. Ad-βgal analyzed by t-test. +++p < 0.001 TGFβ vs. TGFβ + JIB-04 analyzed by ANOVA. Scale bar = 50 μm
