Fig 2: ‘Really interesting new gene’ finger protein 126 (RNF126)-induced mTOR downregulation. (A) Endogenous interaction between RNF126 and mTOR in leukemia cells as determined by immunoprecipitation assay. Normal IgG antibody was used as a negative control. (B) p-mTOR, mTOR or RNF126 protein expression in K562 cells treated with NTS and determined by Western blot. (C) Colocalization between RNF126 and mTOR in NTS-treated K562 leukemia cells. RNF126 (red) or mTOR (green) was stained with anti-RNF126 or anti-mTOR antibodies. Scale bar-10 μm. (D) Downregulation of mTOR in RNF126-overexpressing K562 cells. RNF126 plasmids were transfected into K562 cells and after 24 hours, protein levels were assessed by Western blot. (E) Western blot of enhanced mTOR protein level in siRNF126 knock-down K562 cells. Cycloheximide (CHX, 50 μg/ml) was pre-treated for 1 hour. (F and G) Western blot of mTOR downregulation and leukemia cell growth inhibition in K562 cells transfected with RNF126 wild type (WT) and E3 ligase inactive mutant (MT) (F) MTS assay showing cell viability in K562 cells transfected with RNF126 wild type (WT) and E3 ligase inactive mutant (MT) (G, n = 6). (H and I) RNF126 knock-down affects NTS-induced mTOR downregulation and cell death. RNF126 siRNA was transfected into K562 cells for 48 hours. After then, NTS was treated further 24 hours. Proteins level was evaluated by Western blot using each indicated antibodies (H) and cell viability was measured by MTS assay (I, n = 6).

Fig 3: Translational buffering maintains a largely stable protein expression in 2iL ESCs.a Venn diagram showing the overlap between RNAs, RFPs, and proteins detected in SL, 2iL, or EPI states. RNAs with minimum of 50 reads and RFP with minimum of 25 reads in at least one of the ES conditions were selected and intersected with the 5969 detected proteins. Only uniquely annotated proteins that could be detected in all three conditions (to allow for fold change comparison) were maintained, giving rise to n = 3294 uniquely assigned RFP–RNA–protein IDs. b Scatter plot showing the correlation between fold change in RFP, RNA, and proteins when SL or EPI are compared to 2iL state. Values represent the mean of two highly similar biological replicates. n = 3924 RFP–RNA–protein IDs. R-values represent Pearson correlation coefficients. c Scatter plot showing the correlation between changes in TE and protein expression when SL or EPI are compared to 2iL state. Values represent the mean of two highly similar biological replicates. n = 3924 RFP–RNA–protein IDs. r-values represent Pearson correlation coefficients. d Box plots showing the opposite changes in RNAs and RFPs abundances that result in maintenance of constant protein levels in different states of ESCs and during the SL-to-2iL or 2iL-to-SL transition. Box=25–75th percentile; bar=median; whiskers=5–95th percentile. e Bar plot showing the fold change in RNA-, RFP-, and protein-levels of Rnf126 during 2iL-to-SL transition and in EPI state. f Bar plot showing the distribution of Rnf126 mRNAs in low-density and high-density polysome fractions in different states of ESCs. The qRT-PCR values represent the mean of two highly similar biological replicates. g Western blot analysis of RNF126 in different ESC states. Two biological replicates were used per condition.

Fig 4: Schematic diagram of RNF126-mediated mTOR ubiquitination in NTS-treated leukemia cells. NTS induces activation of RNF126-mediated mTOR ubiquitination and degradation. NTS-induced mTOR degradation affects lysosomal activity, resulting in accumulation of autophagosomes in cells and subsequent apoptosis. NTS-induced antileukemic effects and/or mTOR ubiquitination are inhibited by antioxidant, NAC.

Fig 5: Knockdown of RNF126 leads to decreased replication stress. A, C Western blot analysis showed AZD6738 (1 μM) led to a greater increase in levels of p-RPA2 and γ-H2AX in parental cells, compared to cells with RNF126 knockdown by RNF126 shRNA#1 (A, MCF7 cells; C, MDA-MB-231). B, D Band intensities were quantified and are presented as bar graphs (Two-way ANOVA, p-RPA2, upper panel; γ-H2AX, down panel). E Schematic of DNA fiber analysis. a, red tracks, IdU; b, green tracks, CldU, scale bar,1 μm. F AZD6738 (1 μM) increased the rate of replication initiation, particularly in cells with intact RNF126, compared with cells depleted of RNF126. The frequency of new origins was calculated as the number of green signals (b) divided by the total of green plus red signals (a + b) (One-way ANOVA, MCF7, left panel; MDA-MB-231, right panel) G AZD6738 induced a greater decrease in replication fork speeds in MCF7 (left panel) and MDA-MB-231 (right panel) cells compared with corresponding cells with RNF126 knockdown. The CIdu/Idu ratio was used to determine elongation (One-way ANOVA). H, I The proportion of cells with foci of CDC45 in MCF7 (H) and MDA-MB-231 (I) cells with or without RNF126 knockdown. Cells were treated with AZD6738 (1 μM) for the indicated times and then subjected to immunofluorescence staining. Representative foci of CDC45 are indicated (Paired t-test, scale bar, 5 μm). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. All presented results are from three independent experiments