Fig 2: Protein expression and citrate synthase activity in plantaris muscle in adult offspring sired by control or obese fathers and sedentary or exercised mothers A–C, protein expression of PGC1α (A), Tfam (B) and PHF20 (C). Representative western blots show the quality and signal obtained with the respective antibodies; because they represent one animal, they do not necessarily represent an exact mean of their experimental group. D, citrate synthase activity in plantaris muscle. NS, offspring sired by control diet fathers and sedentary mothers (n = 10); HS, offspring sired by obese fathers and sedentary mothers (n = 10); NE, offspring sired by control diet fathers and exercised mothers (n = 10); HE, offspring sired by obese fathers and exercised mothers (n = 9). Values are presented as mean ± SD. *Paternal obesity effect, P < 0.05. +Paternal obesity vs. Maternal exercise interaction, P < 0.05, followed by a least significant difference test (HS vs. HE).

Fig 3: SIRT1 knockout impacted multiple downstream effectors in aging HSCs.a Venn diagram analysis identified 250 genes that were up in OWT compared to YWT, but were down-regulated in OKO HSC cells. b Realtime RT-PCR validation of key genes identified in a. Geometric averaging of multiple control genes (β-actin, 18S RNA and CD45) was used to normalize data. c Trim26 mRNA in purified HSCs in independent aging cohorts (n = 7 each). d Trim26 and Sirt1 mRNA in spleen and heart using unfractionated cells. e Western blot analysis of MEF cell lysates. f, g Overlap of PHF20 target genes with genes down in OKO over OWT HSCs (f) and 222 annotated genes from 250 genes that were up in OWT vs YWT but down in OKO (g). *P < 0.05; **P < 0.01; ***P < 0.001. n.s. not significant. Error bars represented one standard deviation.

Fig 4: PHF20 inhibition promotes apoptosis in FaDu cells. (A) Western blot assay demonstrated that the expression of MCL1 and cleaved PARP1 decreased in cells with PHF20 knockdown. (B) Quantitative analysis of PHF20, MCL1 and cleaved PARP1 in A. (C) Flow cytometry assay revealed that the proportion of apoptotic cells increased in FaDu cells following PHF20 knockdown compared with that in the control group. (D) Quantitative analysis of apoptotic rates in C. The experiments were performed in triplicate, and unpaired t-test was used for analysis. **P<0.01 and ***P<0.001. PHF20, plant homeodomain finger protein 20; shPHF, lentivirus-mediated shRNA targeting PHF20; Scr, scramble shRNA; PARP1, poly(ADP-ribose) polymerase 1; MCL1, myeloid cell leukemia-1.

Fig 5: PHF20 inhibition suppresses the viability of FaDu cells. (A) The protein expression levels of PHF20 were detected in shPHF and Scr cells by western blot assay. (B) Quantification of the western blotting results in A. (C) The mRNA expression levels of PHF20 were detected in shPHF- and Scr-transfected cells by reverse transcription-quantitative PCR. (D) The viability of cisplatin-sensitive and resistant FaDu cells was detected using the Cell Counting Kit-8 assay. (E) The migration assay demonstrated no significant differences in the cell numbers between the shPHF and Scr groups. Magnification, ×100. Scale bar, 200 µm. (F) Quantitative analysis of the migratory cells in E. (G) The Invasion assay results were similar to those of the cell migration assay. Magnification, ×100. Scale bar, 200 µm. (H) Quantitative analysis of the invasive cells in G. The experiments were performed in triplicate, and unpaired t-test was used for analysis. ***P<0.001. PHF20, plant homeodomain finger protein 20; shPHF, shRNA targeting PHF20; Scr, scramble shRNA.