Fig 2: LEF-10 functionally replaces the prion domain of the yeast Sup35 protein. a Yeast ade1-14 cells expressing LEF-10-Sup35MC were spread on complete (1/4 YPD) medium. [LEF+] strains formed white colonies which were distinguishable from [lef-] strains by the red pigment accumulated through the adenine biosynthetic pathway (top). Both phenotypes could be sustained during cell propagation (middle). [lef-] strains spontaneously generated [LEF+] colonies at a very low frequency (bottom, a colony with the [LEF+] phenotype is indicated by an arrow) and these [LEF+] colonies displayed Ade+ (suppression of ade1-14) phenotype when tested directly on SD-Ade medium. b Comparable characteristics of LEF-10-Sup35MC and full-length Sup35 in yeast. Serial dilutions of [LEF+] and [lef-] strains were spotted on the 1/4 YPD plate (the top panel), or medium lacking adenine (SD-Ade) on which only prion-containing i.e., [PRION+] strains suppressing the stop codon in the yeast ade1-14 allele could grow (the second panel). SDS-resistant aggregates in cell lysates of yeast strains expressing LEF-10-Sup35MC were examined by SDD-AGE (the third panel). The expression levels of full-length Sup35 and LEF-10-Sup35MC were examined by Western blot, probing with a Sup35C-specific antibody (the fourth panel). Endogenous phosphoglycerate kinase 1 (PGK1) was detected with a PGK1-specific antibody and served as a loading control (the bottom panel). [PSI+] and [psi-] strains were used as positive and negative controls

Fig 3: Cavin3-deficient HeLa cells exhibit abolishment of DNA repair.(A) Representative western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. (B) Protein components of the BRCA1 A-complex. Blue-colored circles: proteins downregulated in the label-free quantitative (LFQ) proteomics; yellow-colored circles: proteins not detected in the LFQ proteomics of cavin3 KO cells. (C) Representative western blot analysis of cavin3, BRCA1, pH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1, and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hr chase. Quantitation of protein levels from three independent experiments is presented in Figure 8—figure supplement 1. (D) Representative immunofluorescence images of BRCA1 foci after UV treatment in WT HeLa cells. (E) Percentage of cells with more than five BRCA1 foci, Rap80 foci, and ?H2AX foci in WT and cavin3 KO cells following UV treatment and a 30 min chase. The results are presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (F) WT and cavin3 KO cells untreated or treated with the PARP inhibitor (AZD2461, PARPi) 5 nM for 6 days were subjected to comet assays. The results are presented as the mean ± SEM using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color-coded. Extent Tail Moment was calculated as described in Materials and methods. NS: not significant; **p<0.01, ***p<0.001; ****p<0.0001. Figure 8—source data 1.Raw western data for HeLa WT and cavin3 KO cells time course after UV treatment with molecular weight markers for Figure 8A.Western blot analysis of (A) anti-rabbit cavin3, (B) anti-rabbit CAV1, (C) anti-rabbit BRCA1, (D) anti-rabbit RAD51, and (E) anti-mouse Tubulin antibodies in (1) WT control, (2) WT UV 30 min chase, (3) WT UV 60 min chase, (4) WT UV 120 min chase, (5) WT UV 240 min chase, (6) cavin3 KO control, and (7) cavin3 KO UV 30 min chase, cavin3 KO UV 60 min chase, cavin3 KO UV 120 min chase, and cavin3 KO 240 min chase. Figure 8—source data 2.Raw western data for HeLa WT and cavin3 KO cells untreated or UV treatment for 4 hr with molecular weight markers for Figure 8C.(A) Western blot analysis of anti-rabbit cavin3, (B) anti-rabbit BRCA1, (C) anti-rabbit Rad51 and ?H2AX, (D) anti-rabbit BRCC36, (E) anti-sheep Merit40, (F) anti-rabbit BRCA2, (G) anti-rabbit CAV1, (H) anti-rabbit PKM, (I) anti-rabbit PGK1, and (J) anti-actin antibodies in (1) WT untreated cells, (2) WT + UV treatment and a 4 hr chase, (3) cavin3 KO cells, and (4) cavin3 KO + UV treatment and a chase 4 hr chase time.