Fig. 5: Multi-omic analyses identified HMGN1 as a protein involved in ALT.
From: Telomeres reforged with non-telomeric sequences in mouse embryonic stem cells

a Volcano plot showing the differentially expressed proteins in post-ALT cells (PD800) compared to pre-ALT cells (PD100). Quantitative proteomic analysis was done in biological triplicate for PD800 and duplicate for PD100. The red dot lines indicate 1.5 fold change lines (vertical), and 0.1 of false discovery rate (FDR) line (horizontal). b Heat map showing the differentially expressed proteins of the term chromatin organisation. Colour index indicates z-score of protein expressions and ‘count’ (with regard to histogram) indicates the accumulated number of indicated values represented in the heatmap. c UMAP projection of single-cell RNA-seq data. Each dot represents a different cell. Each plot is colour-coded by identified clusters (top left), expression level of Col4a1 (top right), Gapdh (bottom left), and Hmgn1 (bottom right). d Genes were ranked according to their contributions to differentiation of the PD800 cluster from the others. e Western blot showing HMGN1 expression levels in post-ALT (PD800) and pre-ALT (PD100) cells. Tubulin was used as a loading control. f Representative image of immunostaining of HMGN1 and telomeres. Scale bar, 5 μm. g Quantification of HMGN1 and telomere co-localisation. The dots and bars represent means and confidence intervals for the means, respectively, from ≥200 cells per condition over three independent experiments. P value from a two-tailed unpaired t-test: ***P < 0.0001. h Chromatin immunoprecipitation (ChIP) analysis with HMGN1 antibody. The bars represent the means and SDs from three biologically independent replicates. P value from a two-tailed unpaired t-test: ***P < 0.0001, *P = 0.0130, NS non-significant. i Violin plot showing the TIFs of control and HMGN1 knock-down cells. Each dot represents TIFs in a cell. The dots and bars represent means and confidence intervals for the means, respectively, from ≥200 cells per condition over three independent experiments. P value from a two-tailed unpaired t-test: ***P < 0.0001. j Telomere length quantification with mmqPCR. The bars represent the means and SDs from three biologically independent replicates. P values from a two-tailed unpaired t-test: Region 1 *P = 0.0371, Region 2 *P = 0.0433, Region 3 *P = 0.0466. k TERRA expression quantified by qPCR. All infected cells were post-ALT (PD800). P values from a two-tailed unpaired t-test: Region 1 *P = 0.0116; Region 3 *P = 0.0293. l Snapshot of the mTALT region of RNA-seq. ‘forward’ and ‘reverse’ denote the transcription directions. m DRIP-qPCR quantified with specific primers. The bars represent the means and SDs from five biologically independent replicates for no RNaseH1-treated samples and three for RNaseH1-treated samples. All infected cells were post-ALT (PD800). P values from a two-tailed unpaired t-test: Region 1 NS, non-significant, ***P = 0.0009, **P = 0.0040; Region 2 *P = 0.0331, ***P = 0.0009, **P = 0.0079; Region 3 *P = 0.0332, ***P = 0.0013, **P = 0.0050. n Representative images of telomere fragility. Scale bar, 1 μm. o Telomere fragility was quantified from chromosome orientation (CO) FISH. The bars represent the means and SDs from ≥30 cells per condition over three independent experiments. P value from a two-tailed unpaired t-test: ***P = 0.0002. Regions 1–3 denote specific regions inside the mTALT sequence. Source data are provided as a Source Data file.