Fig 2: RUNX3 sequentially recruits TrxG and PcG complexes. a Yeast two-hybrid screening using Gal4-BRD2 (aa 450–802) as bait identified RNF2 and MLL5 as BRD2-binding proteins (see STAR methods). DDO = SD-Leu/-Trp, DDO/X/A = SD-Leu/-Trp/X-a-gal/ABA, QDO/X/A = SD-Leu/-Trp/-His/-Ade/X-a-gal/ ABA medium. Selective colonies were identified by DNA sequencing. b HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points, and the time-dependent interactions between RUNX3, BRD2, Cyclin D1, MLL1, MLL5, RNF2, BMI1, EZH2, EED, and HDAC4 were measured by IP and IB. c PLA showing RUNX3-MLL1 and RUNX3-MLL5 at the indicated time points after serum stimulation. Green fluorescence indicates association of the indicated proteins. F-actin was stained (red) to visualize the cytoplasmic compartment. d Microscopy images of transgenic fly eyes. Lozenge is a Drosophila homolog of the RUNX genes. Glass multimer reporter (GMR)-Gal4 promotes eye-specific expression of UAS-inserted genes. GMR-driven Lozenge overexpression (GMR-Gal4/ + ;UAS-Lozenge (lz) ? GMR > Lz) or GMR-driven Trithorax (Trx) overexpression (GMR-Gal4/ + ; TrxG14137 ? GMR > Trx) conferred weak rough phenotypes. However, GMR-driven overexpression of both Lz and Trx (GMR > Lz + Trx) resulted in a severe defective eye phenotype with loss of external ommatidial facets. e HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. The binding of RUNX3, BRD2, MLL1, MLL5, CDK4, RNF2, Cyclin D1, HDAC4, EZH2, H2A-K119-Ub, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. f Schematic illustration of the R-point transition. At 1 h after serum stimulation, RUNX3 associates with various proteins, including p300, BRD2, H4K12-ac, SWI/SNF, TFIID, and MLL1/5, to form Rpa-RX3-AC. Between 2 and 4 h after serum stimulation, Rpa-RX3-AC interacts with PRC1–CyclinD1–HDAC4 to from a transient complex, Rpa-RX3-TR. Subsequently, Rpa-RX3-TR is destroyed (at 4 h) to form Rpa-RX3-RE (at 8 h)

Fig 3: The fermented product induces IL-8 transcription. HaCaT cells were treated for 24 h as indicated; (A,B) the levels of IL-8 transcript were assessed by real-time PCR. (C) NF-kB protein expression was detected by western blot from the cytosolic and nuclear extracts of healthy cells. Actin and TBP expression were used as loading control. Bands were quantified relative to loading control, and the translocation from cytosol to the nuclear compartment was evaluated as the ratio of nuclear vs. cytosolic expression. (D) A representative blot is shown, and the whole blots are displayed in Supplementary Materials Figure S1. The data represent the means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the control (ctrl); §§§ p < 0.001. IL-8, interleukin-8; PCR, polymerase chain reaction; NF-kB, nuclear factor kappa B; TBP, TATA-Box Binding Protein; SEM, standard error; GOS, galactooligosaccharides; ctrl, control; perm, permeate; ferm, fermented product; LPS, lipopolysaccharide from Escherichia coli O111:B4.

Fig 4: GOS and whey derivatives induce the transcription of respiratory subunits and promote FOXO-1 translocation. HaCaT cells were treated for 24 h as indicated, and the levels of mitochondrial transcripts were assessed by real-time PCR (A,B). FOXO-1 protein expression was detected by western blot from nuclear and mitochondrial extracts. VDAC and TBP expression was used as loading control. (C) Bands were quantified relative to loading control, and the translocation from the mitochondrial to the nuclear compartment was evaluated as the ratio of mitochondrial vs. nuclear expression. (D) A representative blot is shown, the same membrane displayed in Figure 1, and the whole blots are available in Supplementary Materials Figure S1. The data represent the means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the control (ctrl); §§§ p < 0.001. FOXO-1, Forkhead Box O1; VDAC, voltage-dependent anion channel; mitoc COX II, mitochondrial cytochrome c oxidase subunit 2; MT-ATP6, mitochondrial adenosine 5'-triphosphate (ATP) synthase F0 subunit 6.

Fig 5: Arsenite and proteasome inhibitor MG132 induce UBC gene expression and promote nuclear accumulation of HSF1, HSF2 and Nrf2 transcription factors. (A) HeLa cells were treated with NaAsO2 (80 µm) or MG132 (20 µm) over an 8-h time course (as indicated). UBC mRNA levels were determined by RT-qPCR, normalized to GAPDH levels and expressed as fold increase relative to control cells (NT/DMSO). (B) HeLa cells treated for 8 h with different concentrations of NaAsO2 (as indicated) were analyzed as in (A). UBC mRNA level is shown as fold increase versus the untreated sample (NT). (C) Cell viability of HeLa cells exposed to NaAsO2 or MG132 for the indicated times (n = 3 each) were evaluated by the MTS assay and shown as a percentage with respect to the untreated-cell value (time 0 h), set equal to 100. (D) Cells treated with NaAsO2 or MG132 for 4 and 8 h and untreated cells (NT) were subjected to the proteasome activity assay (n = 4). (E) Nuclear levels of HSF1, HSF2 and Nrf2 transcription factors upon cell treatment with stressors for the indicated times were analyzed by western immunoblotting of nuclear fractions, with anti-HSF1, anti-HSF2 and anti-Nrf2 specific antibodies. TFIID and Sp3 were used as nuclear loading controls. Arrows indicate the detected protein. (F) HSF1, HSF2 and Nrf2 mRNA levels in cells treated for 8 h with NaAsO2 or MG132 (n = 3 each) were measured by RT-qPCR and expressed as a fold change relative to NT cells. All data are expressed as means ± SEM of the indicated number of samples. Asterisks denote statistical significance, calculated by one-way ANOVA, versus control as specified or indicated by bars; *P < 0.05; **P < 0.01; ***, ###P < 0.001; n.s., not significant.