EMBO/SourceData huggingface dataset:
Figure 2C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen. Data information: In (C rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen. Data information: In D) rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, red, and blue arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).
Figure 3Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). homboid substrates that are uncleaved, are marked by black, rrows, ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, re marked by black, red, and blue arrows, espectively. ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.
Figure 4A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.B Western blot analysis to detect HybA cleavage in S. sonneiΔrhom7 by wild-type (+) or inactive (S201A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (-) HybB. HybA was expressed from its native locus or a plasmid (pHybA). HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.C Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (-) HybB. mean ± S.D. of three experiments. *, p<0.05; ****, p<0.0001 (One-way ANOVA).D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.
Figure 5B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively.E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.
Figure 6A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, , are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI. Rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively.D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.
Figure 7A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.
Figure 2(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.
Figure 3(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(B) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16-F10 tumor cells cultured with the indicated cytokine(s) (10 ng/mL for all) for 0, 3, 6, or 9 hours; β-actin was used as a loading control.(D) CRISPR/Cas9 screen showing enriched genes from sgRnf31 B16-F10 tumor cells after successive treatment with combined TNF/IFN-γ, as described in (C); red genes indicate overall genes with p < 0.0001. Significance assessed using MAGeCK test.(G) PI staining of sgControl, sgRnf31, sgRnf31/Stat1, sgRnf31/Casp8, sgRnf31/Bid, or sgRnf31/Rela B16-F10 tumor cells treated with combined TNF/IFN-γ for 24 hours; *p < 0.05, ****p < 0.0001, asterisk(s) indicated group vs TNF/IFN-γ-treated sgRnf31 group, Unpaired t-test for n > 3 biological replicates. Pooled data are represented as mean ± SEM.
Figure 4(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.
Figure 5(B) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.(C) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.
Figure 1(B) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CGA inhibitory pair (red) and the CGC optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).(C) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CCG inhibitory pair (red) and the CGC-CCA optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).
Figure 2(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).(B) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).(C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).
Figure 3(A) Meta-analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site.(B) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair (red) compared to their corresponding optimal pair (green). P-values were determined by Student's t-test between each inhibitory and the corresponding optimal pair (** p < 0.01, n=2).(C) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX/ANS (blue) and CHX/TIG (black) (left) to their corresponding optimal pairs with the same antibiotic combination (right). No significant difference found (n.s.: p=0.9961, n=2) between CHX-TIG and CHX-ANS.
Figure 1E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.
Figure 2D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM. F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm.H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm. I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM. J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm. K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.
Figure 3H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).
Figure 4A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm. C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm.
Figure 5B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm. C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus. F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm. G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E. I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test). J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse). K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice. L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.
Figure 6A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm. E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR). F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO). G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm. I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO). J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm. L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm. M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).
Figure 7D, Average time animals needed to reach the platform per day. Note that KO mice showed increased path length and latency on days 4 and 5. E, Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase.
Figure 2(A) MCF-7 cells were co-transfected with either MIR-CNT (control plasmid) or MIR376A and GFP-LC3 plasmid, and GFP-LC3 dot formation was analyzed. White arrows indicate clusters of the GFP-LC3 dots in cells. (B) Quantification of the experiments in A. MIR376A overexpression, but not MIR-CNT expression, blocked starvation (STV)-induced autophagy (mean ± S.D. of independent experiments, n = 4, ***p<0,01). NON STV, non-starved(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.(D) MIR376A blocked starvation induced SQSTM1 degradation in MCF-7 cells. ACTB was used as a loading control. SQSTM1/ACTIN densitometric ratios were marked.
Figure 3(A) MIR376A blocked GFP-LC3 dot formation under starvation condition.(A) MIR376A blocked GFP-LC3 dot formation under starvation condition. (B) Quantitative analysis of experiments in A (mean ± S.D. of independent experiments, n = 3, ***p<0,01).(C) Overexpression of MIR376A resulted in decreased autophagic flux in Huh-7 cells. Starvation-induced conversion of LC3-I to LC3-II was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.
Figure 4(C and D) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA (MIR-CONT) or MIR376A overexpressing cells under non-starved (NON STV) or starved (STV) condition (mean ± S.D. of independent experiments, n = 3, *p<0,05).(E and F) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA (MIR-CONT) or MIR376A overexpression. ACTB was used as a loading control. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.
Figure 5(A and B) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF-7 cells transfected with control antagomirs (CNT-Ant) or antagomir-376a (Ant-376a) (mean ± S.D. of independent experiments, n = 3, **p<0,03 for ATG4C, and n = 3, **p<0,03 for BECN1). Results were expressed as fold changes against GAPDH mRNA levels.(C and D) ATG4C and BECN1 protein levels were increased following antagomir-376a (Ant-376a) transfection. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.
Figure 6(C and D) Normalized luciferase activity measuremets of lysates from 293T cells that were co-transfected with either MIR-CNT or MIR376A plasmids together with the wild-type or mutant luciferase constructs (mean ± S.D. of independent experiments, n = 3 for ATG4C tests, *p<0,05 and n = 5 for BECN1 tests, ***p<0,01).
Figure 7(A) Blockage of endogenous MIR376A by Ant-376a, but not CNT-Ant further stimulated starvation (STV)-activated LC3-I to LC3-II conversion in MCF-7 cells. ACTB was used as a loading control. LC3-II/LC3-I densitometric ratios were marked.(B) Ant-376a, but not CNT-Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF-7 cells. SQSTM1/ACTB ratios were marked.
Figure 8 MIR376A and MIR376B level changes in response to starvation stress. (A and B) TaqMan quantitative PCR (qPCR) analysis of endogenous MIR376A (A) and MIR376B (B) levels under control (CNT, no starvation) or starvation (STV, 1 to 8 hours) conditions. TaqMan qPCR data was normalized using U6 small nuclear 1 (RNU6-1) (U6) mRNA levels (mean ± S.D. of independent experiments, n = 5 for both A and B, ***p<0,01).
Figure 1Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.
Figure 3Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.Rmi1, a component of the Sgs1-Top3-Rmi1 (STR) complex, is hyper-phosphorylated on S/T-Q motifs in rad9Δ cells.