Priority Research Paper Advance Articles

Acid ceramidase promotes senescent cell survival

ASAH1 is highly expressed in senescent cells. (A) Senescence-associated β-galactosidase (SA-βGal) 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.

Figure 1. ASAH1 is highly expressed in senescent cells. (A) Senescence-associated β-galactosidase (SA-βGal) 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.