Fig 2: Single-cell RNA-seq analysis identifies distinct cell clusters with unique expression profiles of MRPs and shleterin genes associated with senescence. (A) Identification of highly variable features in scRNA-seq data derived from aging mouse dermal fibroblasts (GSE111136). (B) Distribution of p-values for each PC with a uniform distribution (dashed line) showing significant PCs. (C) Uniform manifold approximation and projection (UMAP) plots depicting cells of different Seurat-defined cell clusters (left) and ages (right). (D) Stacked bar charts showing the number and proportion of cells assigned to Seurat-defined cell clusters. (E) Expression heatmaps of the top 10 genes for each sample (young and old) in C3 (top) and C4 cells (bottom) only. (F) Dot plot showing top GO terms enriched in each cell group. (G) Expression heatmaps and (H) mean expression (±SD) of the 47 mouse SA-MRPs, 15 SA-MRP genes, shelterin complex, Acd, and senescence markers (Cdkn1a for p21 and Cdkn2a for p16). Kruskal–Wallis chi-squared p-values (p) are shown. N refers to the number of genes analyzed. Of the 51 SA-MRPs, Mrps16, Mrps37, Mrps30, and Mrps27 are missing in the filtered dataset.

Fig 3: SA-MRP deregulation plays a role as an upstream regulator of TPP1 suppression. (A,B) HDFs (DT2) were transfected with siRNAs against targets (TPP1 and POT1) for 4 days. (A) Western blots. Representative blot images and their quantified values are shown. (B) Messenger RNA levels by qPCR 4 days after individual knockdown of TPP1 and POT1. **, p < 0.01 vs. NC by Student’s t-test. (C–G) HDFs (DT2) were transfected with siRNAs against target MRPs for 4 days. (C) Western blots. Representative blot images and their quantified values are shown. (D) Messenger RNA levels (qPCR) 4 days after individual knockdown of MRPS9, MRPS15, and MRPS31. **, p < 0.01 and *, p < 0.05 vs. NC by Student’s t-test. (E) Relative telomere length. (F) Telomere dysfunction-induced focus (TIF), a co-localized focus of telomere (green) and 53BP1 (red) was visualized by IF–FISH, as described in Materials and Methods. DAPI (blue) staining was used to visualize nuclei. Quantifications of the merged images are presented in Supplementary Figure S4B. (G) Western blot. Representative blot images and their quantified values are shown. (H,I) HDF (DT2) was exposed to CAP (200 µg/mL) for the indicated periods. (H) Western blots. Representative blot images and their quantified values are shown. (I) Quantitative RT-PCR. **, p < 0.01 and *, p < 0.05 vs. NC by Student’s t-test. (J) Western blot analysis after HDF (M stage, PD63 and DT3) was transfected with the pCMV6-ACD-AC-GFP plasmid for 2 days. Representative blot images and their quantified values are shown. (A,C,H) arrow indicated POT1 band.