Extended Data Fig. 1: Up-regulation of Cbs and Cth upon Lmna loss.
From: Lamin A/C-regulated cysteine catabolic flux modulates stem cell fate through epigenome reprogramming
a, Quantification of the distance of Cbs and Cth to nuclear periphery in individual nuclei of Lmna+/+ and Lmna-/- mES cells. Cbs: Lmna+/+ (n = 105) and Lmna-/- (n = 161); Cth: Lmna+/+ (n = 106) and Lmna-/- (n = 103). b, Integrative Genomics Viewer (IGV) tracks of ATAC-seq and RNA-Seq of control (grey), and Lmna-/- mES cells (red) at the Cbs and Cth locus. Significantly increased peaks are highlighted in beige boxes. c, Relative fold changes in ATAC-seq signal at the transcription start site (TSS) and distal regions of Cbs and Cth in Lmna-/- mES cells compared to control mES cells. Differential accessibility was calculated in DESeq2 using two-sided Wald test. n = 3 biological replicates. d, RNA-seq analysis showing RPKM values of Cbs and Cth in Lmna-/- and control mES cells. n = 3 biological replicates. Differential expression was calculated in DESeq2 using two-sided Wald test. e, CTH (left) and CBS (right) enzymatic activity in control and Lmna-/- mES cells. n = 3 biological replicates. Purified CBS and CTH protein served as a positive control. f, g, Immunoblot analysis of key enzymes involved in the one-carbon metabolism in control and Lmna-/- mES cells (f), and quantification of protein relative fold changes (g). n = 3 biological replicates. h, i, Representative immunoblot for SP1 (h) and ATF4 (i) in chromatin-bound fraction (CBF) and total protein extract of control and Lmna-/- mES cells. Lamin B1 and α-Tubulin served as a loading control. j, Relative mRNA expression of Cbs and Cth in WT mES cells treated with DMSO or the Ezh1/2 inhibitor UNC1999. n = 4 biological replicates. k, ChIP–qPCR for H3K27me3 at the Cth and Cbs promoter in control and Lmna-/- mES cells normalized to input. n = 4 biological replicates. l, ChIP–qPCR for H3K27me3 and SP1 in mES cells treated with DMSO or UNC1999. n = 3 biological replicates. Differences in a, e, g, j–l were assessed using an unpaired two-tailed Student’s t-test. Data are presented as mean ± SD.
