EMBO/SourceData huggingface dataset:
Figure 3Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting.Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).
Figure 4Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).FLAG-immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG-expressing cells after siRNA-mediated down regulation of ACAD9 and pulse radiolabeling. SDS-PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3 and ND4L were normalized to ATP6 (total) or isolation efficiency (eluate). Total 2 %, Eluate 100 %. Error bars indicate SEM from three biological replicates; **p < 0.01; unpaired t-test.After C12orf62 down regulation, [35S]methionine labelling was performed. During labeling mitochondrial translation was inhibited with puromycin (pur) (2 µg/ml). MITRAC15FLAG interacting nascent chains were isolated by FLAG-immunoprecipitation from cell lysates. Eluates were subjected to SDS-PAGE, analyzed by digital autoradiography. Total 2 %, MITRAC15FLAG eluates 100 % (in case of C12ORF62FLAG, 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 is presented in a magnification. F1-F3 mark nascent chains of COX1 [18].HEK293T cells were pulse labelled with [35S]methionine in the presence of puromycin (2 µg/ml). During puromycin treatment, cells were incubated for 5 min and 10 min in medium lacking [35S]methionine. Anti-FLAG immunoisolations were performed as described and eluates analyzed as described in Fig. 4C. Total 1 %, Eluates 100 %. The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % (error bars indicate SEM from three biological replicates; *p < 0,05; paired t-test). Red asterisks, accumulating nascent chains of ND2.
Figure 5Radiolabeling with [35S]methionine, anti-FLAG immunoisolation and sample analyses were performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.Depletion of ACAD9 by siRNA was performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.
Figure 1(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments.(D, E) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT, snf1Δ or snf1Δmig1Δ cells (D). Protein extracts were probed with an anti-GFP antibody. Mcm2 was applied as a loading control. Three independent experiments were carried out and the relative fold changes were measured by Image J (E). Error bars represent standard deviations (SD) from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05; **0.001< P-values <0.01).
Figure 2(C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above.(D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A.(F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.
Figure 3(C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607.(E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.
Figure 4mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU. The experiments were done as in Fig 2.(E, F) snf1Δ shows less RPA on chromatin, which can be restored by mig1Δ. Cells were collected and fractionated into chromatin-bound (Chromatin) and non-chromatin-bound fractions (non-Chromatin) followed by immunoblotting. Tubulin and Orc2 were applied as controls for non-chromatin and chromatin fractions, respectively. The signals were quantified by Image J and quantifications were shown in (F). Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).
Figure 5Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C)Phospho-mimetic mig1-2D bypasses the role of SNF1 in RPA recruitment (D). (E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel).
Figure 6(C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats.(D) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ. The experiments were done as in Fig 1. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).
Figure 7(B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A.(D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control.(E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A.
Figure 1a, b. The amount of FDP-lysine, which was major protein-bound acrolein, in the sera of healthy controls and PD patients was determined by an ELISA (a. all patients, (control n= 22; PD n=94) b. staged by the H&Y scale, a five-steps index of PD severity. Larger number indicates more severe condition. n=94; H&Y stage I (n=18), stage II (n=42) stage III (n=23), stage IV (n=7), and stage V (n=4).
Figure 2SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right).e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.h. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.
Figure 3d. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. (upper) Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.
Figure 4a. Cells were transfected with the TMEM55B-myc-DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h. Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies.b. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).c. SH-SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed, subjected to Phos-tag PAGE, and immunoblotted with an anti-JIP4 antibody. d. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI (Jak3 i VI) for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells lysates were subjected to Phos-tag PAGE and immunoblotted with an anti-JIP4 antibody.e. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).SH-SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h. Cell lysates were subjected to Phos-tag PAGE and immunoblotted with anti-JIP4 antibody (f, left). CaMK2G knockdown efficiency was confirmed by qRT-PCR.; ***p <0.001, n=3 technical replicates. Values are mean ± SD. At least three experiments were replicated (f, right).j. His6-tagged recombinant truncated JIP4 (100-300 aa) was purified from E. coli and incubated with recombinant CaMK2G with/without Ca2+ and calmodulin at 30°C　for 1 h. The reactant was subjected to Phos-tag PAGE and immunoblotted with anti-His antibody. Jak3 inhibitor VI was pre-reacted with CaMK2G for 30 min.
Figure 5c. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2 h. Cell lysates were immunoprecipitated with anti-RFP magnetic beads and immunoblotted with the indicated antibodies.f. SH-SY5Y cells transfected with GFP or GFP-ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies.
Figure 6Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h (a). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI (Jak3 i VI) for 2 h (b). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.d. (upper) SH-SY5Y cells were pretreated with 10 µM BAPTA-AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.e. SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were lysed, subjected to Phos-tag PAGE, and immunoblotted with anti-JIP4 antibody.f. (left) SH-SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were fixed and stained with anti-γ-tubulin (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (right) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ***
Figure 7b. Parental and JIP4 KO SH-SY5Y cells were treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. c. SH-SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay.
Figure 1Expression of ERα in control (shCtrl) and knockdown (shERα) BM67 cells was determined by western blotting (β-actin served as a loading control). Gene expression was determined using polysome-profiling which quantifies both total mRNA and efficiently translated polysome-associated mRNA (n = 3).Distributions (quantified by kernel density estimation) of p-values for differential expression (shERα vs. shCtrl BM67 cells) using data from polysome-associated mRNA (orange), cytosolic mRNA (purple) or from analysis of changes in translational efficiency (i.e. changes in polysome-associated mRNA not paralleled by changes in cytosolic mRNA) leading to altered protein levels (red; a higher density of low p-values indicates a higher frequency of changes in gene expression).Scatterplot of polysome-associated mRNA vs. cytosolic mRNA log2 fold-changes (shERα vs. shCtrl). Areas of the plot are colored according to the density of data points (genes; dark blue corresponds to areas with many genesSame plot as in (B) but including a blue density of p-values from the analysis of translational offsetting.A scatter plot similar to (C) but where genes are colored according to their mode of regulation derived from anota2seq analysis. A relaxed threshold (p<0.05) was used to identify a set of transcripts regulated via translation, which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting (FDR<0.1). Confidence ellipses (level 0.7) are overlaid for each mode of regulation. A bar graph indicates the number of mRNAs regulated via each mode.
Figure 2Targets from each mode of regulation (cf. Fig 1E) were selected for validation by Nanostring. Shown is a scatterplot of Nanostring quantification of polysome-associated mRNA vs. cytosolic mRNA log2 fold-change (shERα vs. shCtrl; n = 3). Genes are colored according to their identified mode of regulation (i.e. from Fig 1E). Confidence ellipses (based on Nanostring data, level 0.7) are overlaid for each mode of regulation. Selected genes from each mode of regulation are indicated.Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications. β-actin served as loading control. Graphs represent mean ± standard deviation (n = 3-5 as indicated). Protein expression between groups were compared using two-sided paired Student's t tests.Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).
Figure 3nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries.Boxplots for offset and non-offset mRNAs comparing 5' UTR weighted lengths (log2 scale), GC content (%), free energy (log10 scale, kcal/mole). In boxplots, the solid horizontal middle line is the median; the lower and upper box limits correspond to the first and third quartiles; the whiskers extend from the box limits to the most extreme value but not further than +/- 1.5 x the inter-quartile range (IQR) from the box limits. The notches extend to 1.58 x the IQR / √n where n is the number of data points. Data beyond the end of the whiskers are plotted as points. Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between translationally offset and non-offset mRNAs. Number of transcripts harboring at least one (dark gray) or no (light gray) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one-sided Fisher's exact tests. A global Bonferroni adjustment was applied on p-values. Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells; the numbers of transcripts in each group are indicated in the figure.
Figure 4Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq. Down- and up-regulated miRNAs (FDR<0.2 and fold-change > 1.25) are colored in blue and red, respectively.Validation of miRNA expression using qPCR. Normalized expression (mean ± standard deviation; n = 3) (B) and expression fold change (shERα vs. shCtrl) obtained from small RNAseq vs. qPCR (C).Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells (mean ± standard deviation; n = 3).Assessment of the relationship between presence of miRNA target sites and translational offsetting. (E) For visualization, mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome-associated mRNA obtain an angle of ~45 degrees, whereas offset mRNAs are assigned an angle of ~0 degrees. (F) The distributions (quantified by kernel density estimation) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion, which were also targets of each suppressed miRNAs separately (blue lines); and the set of background transcripts (mRNAs induced upon ERα depletion but not targets of suppressed miRNAs; black). (G) For such subsets of miRNA targets (i.e. from F), the relative number of offset and non-offset mRNAs (as determined by anota2seq analysis) was also compared to the background (same as in F) using Fisher's exact test; and the resulting adjusted p-values (FDRs; across all assessed miRNAs) were visualized as a histogram. (H-I) A similar assessment as in (F-G) but for suppressed mRNAs and induced miRNAs. (J) A similar assessment as in (H) but for randomly selected groups of 10 miRNAs (gray lines) compared to background (black) and targets of all induced miRNA (red; i.e. from H).
Figure 6Immunoblotting of BM67 cell extract for ELP3 after stable shRNA-mediated ERα knockdown in BM67 cells. Graphs represent mean ± standard deviation of n=3.qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5.qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=4.Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector, Elp3-null BM67 cells and Elp3-null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site. Graphs represent mean ± standard deviation of n=3-4.
Figure 7Boxplots (plotted as in Fig 3B) of gene expression of ELP3, ALKBH8 and CTU2 upon treatment with 17β-E2 and/or ICI-182780 in MCF7 cells (extracted from (Wardell et al, 2012; Data Ref: McDonnell et al, 2012)). Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between conditions (n = 10) and a global Bonferroni adjustment was applied on p-values.Immunoblotting of MCF7 cell extracts. MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours and then treated with 1 nM E2 and/or 100 nM ICI-182780 for 24 hours. Graphs represent mean ± SD of n=5 experiments. Data were analyzed using two-sided paired Student's t tests.ChIP-seq reveals ER binding sites proximal to the ELP3 gene. Each track represents a distinct ChIP-seq dataset.Immunoblotting of MCF-7 cell extract for indicated proteins following deletion of ELP3 gene by CRISPR-Cas9 technology.Quantification of mcm5S2-U, mcm5U and cm5U levels in ELP3-null MCF-7 cells and in MCF-7 cells treated with 100 nM ICI-182780 for 72 hours versus their respective controls by liquid chromatography-mass spectrometry (LC-MS) analysis. The MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours prior to ICI-182780 treatment. Graphs represent mean ± SD of n=4. Data were analyzed using two-sided paired Student's t tests. Vh, Vehicle; ICI, ICI-182780.
Figure 1G. Immunostain for Ki67 in wildtype and lymphoma-bearing spleen. Representative images are shown at left. The proportion of nuclei positive for Ki67 (proliferation index) is quantified at right for n=3 spleens(T-test p=0.00002331).
Figure 2F, G (F) PCR for Cγ1-Cre and wildtype Cγ1 alleles and (G), wildtype, floxed or excised Blimp1 alleles in DNA from wildtype (wt) control C57bl/6J cells and purified T-cells or CD45.2+ cells from mouse with lymphoma (T1, T2). PCR data were confirmed in at least 3 independent tests. Lad. = ladder.
Figure 5F. Western blot for c-MYC in whole spleen lysates from control C57bl/6J and mPTCL mice. Quantification of c-MYC expression normalized to vinculin housekeeper gene levels (n=6). T-test p=0.0007. Bars represent SEM.
Figure 6B. Western blot for phosphorylated and total ZAP70, AKT, S6 and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.H. Western blot for γH2AX, total H2AX, CHK1 and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left) and quantification was performed on n=6 at right. T-test p=0.0798 (CHK1; not statistically significant, ns), p=8.69E-05 (RPA32), p=2.11E-03 (γH2AX). ***p<0.001.
Figure 7A. Sanger screen IC50 drug sensitivity data (IC50 values) for DDR inhibitors targeting ATR, CHK1/2 and Wee1 in T-ALL, PTCL (DEL, KARPAS-299, SUP-M2) and pan-cancer cell lines. ANOVA p=0.413 (AZD6738, ATR inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR inhibitor) and 0.282 (AZD1775, Wee1 inhibitor). Bars represent SEM of biological replicates.B. PTCL (red) and T-ALL (yellow) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo. Average IC50 values from three experiments are plotted. Bars represent SEM.F. Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 (ATR; n=4) relative to untreated mPTCL spleens taken at endpoint (n=6). T-test p=2.70E-06. PC9 cells dosed in vitro 24h with 1 µM aphidicolin (Aph; an inhibitor of DNA replication) or with AZD6738 (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001