Fig 2: ASAH1 is highly expressed in senescent cells. (A) Senescence-associated ß-galactosidase (SA-ßGal) staining of WI-38 fibroblasts that were proliferating (P, PDL25) or were rendered senescent (S, PDL52) by extended culture or by exposure to ionizing radiation (IR, 10 Gy) and assayed 10 days later. (B) Western blot analysis of senescence marker proteins p16 and p53 in cells prepared as described in (A). The levels of loading control HSP90 were also assessed. (C) Left, Venn diagram showing commonly upregulated mRNAs (RNA-sequencing data) and proteins obtained by mass spec (MS) analysis (full list provided as Supplementary Table 1) in replicative senescence (S) and IR-induced senescence relative to proliferating (P) fibroblasts. Right, partial list of the shared mRNAs and proteins upregulated in senescent cells. (D, E) In cells that were processed as in (A), the steady-state levels of p21 and ASAH1 mRNAs were quantified by RT-qPCR analysis (mRNA levels were normalized to GAPDH mRNA levels), and the levels of ASAH1 were assessed by Western blot analysis (loading control protein GAPDH was included). Data in (D) are the means ±S.D. of three biological replicates; data in (A, B, E) are representative of three biological replicates. RNA-seq is provided in GSE85771.

Fig 3: Silencing ASAH1 represses senescence. (A) Pre-senescent cells were transfected with either control or ASAH1 siRNA; 48 h after transfection cells were harvested and whole-cell lysates prepared for Western blot analysis to examine the levels of ASAH1, p16, p21, p53, and loading control GAPDH. (B, C) Detection of the senescent marker SA-ßGal using traditional SA-ßGal staining (B) and SPiDER-ßGal (C) in the populations described in (A). (D) Metabolomic analysis of the levels of ceramides, fatty acids, and sphingomyelin after silencing ASAH1. Dashed lines indicate p < 0.05. The x-axis indicates the categories and individual metabolites. Red bars indicate a log odds ratio (OR) > 1: an increased log odds of a higher metabolite concentration in the ASAH1 group relative to the comparison group (Control siRNA). Blue bars indicate a log odds ratio (OR) < 1: a decreased log odds of a higher metabolite concentration in the ASAH1 group relative to the comparison group (Control siRNA). Significant metabolites are highlighted in the table below the graph.

Fig 4: ASAH1 mRNA stability and translation in senescent cells. (A) RT-qPCR analysis of the levels of ASAH1 and GAPDH pre-mRNAs using total RNA from proliferating (P) and senescent (S) cells upon replicative exhaustion and normalized to GAPDH mRNA levels. (B) Proliferating and senescent cells were treated with Actinomycin D for the times indicated to block transcription; total RNA was then isolated, and RT-qPCR analysis was performed to assess the levels of ASAH1 mRNA. The labile MYC mRNA and the stable GAPDH mRNA were included as control transcripts. Discontinuous line indicates 50% of the original levels of mRNAs at time 0. (C) Cytoplasmic lysates obtained from P and S cells were fractionated through sucrose gradients to assess global polysome distribution profiles; ‘Unbound RNA’ fractions, fractions containing small ribosomal subunits (‘40S’), large ribosomal subunits (‘60S’), and monosomes (‘80S’), as well as polysomes of low and high molecular weight (LMWP and HMWP) are indicated. The relative distribution of ASAH1 mRNA and housekeeping ACTB (ß-actin) mRNA was studied by RT-qPCR analysis of RNA in each of 12 gradient fractions. Data in (A) and (B) represent the means and S.D. from three independent experiments; data in C are representative of three independent experiments.

Fig 5: Altered ASAH1-related metabolites in senescence. (A) Schematic representation of the metabolic pathway of ceramides conversion to sphingosine and fatty acids by ASAH1. Ceramides are also converted to sphingomyelin during sphingolipid biosynthesis or generated during sphingolipid recycling. (B, C) Metabolomic analysis of the levels of ceramides, fatty acids, and sphingomyelin during replicative (B) or IR-induced (C) senescence. Dashed lines indicate p < 0.05. The x-axis indicates the categories and individual metabolites. Red bars indicate a log odds ratio (OR) > 1: an increased log odds of a higher metabolite concentration in the Replicatively senescent (B) or IR-induced senescent (C) group relative to the comparison group (Proliferating). Blue bars indicate a log odds ratio (OR) < 1: a decreased log odds of a higher metabolite concentration in the Replicatively senescent (B) or IR-induced senescent (C) group relative to the comparison group (Proliferating). Significant metabolites are highlighted in the table below each graph.