EMBO/SourceData huggingface dataset:
Figure 4B. Representative immunofluorescence FISH (IF-FISH) images to measure CENP-B intensity at specific chromosome in untreated cells. Scale bar represents 5 µm.C. Bar plot showing the normalized CENP-B fluorescence intensity at every chromosome over the mean on each metaphase spread (n > 37 per chromosome) ± SEM. Acrocentric chromosomes 13, 14, 21 and 22 were not analyzed and are marked by a line. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.D. Scatter plot showing a significant negative correlation between the mean of CENP-B IF-FISH signal (n > 37) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence band.E-F. Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP-C binding following CUT&RUN, sequencing and centromere mapping. Cells were treated for 6 h with IAA to deplete CENP-A. Error bars represent the SEM of three independent experiments and the dashed line represents the mean. Acrocentric chromosomes 13-14, 21 and 22 were marked by a line. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001.G. Scatter plot showing a significant positive correlation between the mean of CENP-C reads (n = 3) and CENP-B reads (n = 3) (r = Spearman rank coefficient). Data from chr 1, 5 and 19 were excluded from the analysis in addition to chr 13, 14, 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95% confidence interval.H. Scatter plot showing a significant negative correlation between the mean of CENP-C reads (n = 3) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. The lines represent linear regression with a 95% confidence interval.
Figure 5B. Representative image of centromere 11 (red) and centromere X (green) FISH on RPE-1 CENP-AAID CENP-B KO mutant in control (top) or IAA treated condition (bottom). Scale bar represents 5 µm. Bar graphs show automatic FISH quantification of chromosome mis-segregation in the indicated cell lines with and without IAA addition for 24 h. Error bars represent the SEM of three independent experiments (n> 200 cells per experiment). Dunnett's multiple comparisons test was used to compare conditions. Grey lines separate independent experiments. Fold changes between grey and red bars are also indicated.C. Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound. D. Representative image of centromere 17 (red) and centromere X (green) FISH on RPE-1 cells treated with the BAL27862 compound. Scale bar represents 5 µm. E. Bars represent the frequency of chromosome mis-segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration. Error bars represent the SEM of three independent experiments, dashed line represents the mean. n > 40 micronuclei were analyzed. Dunnett's multiple comparisons test, *: p<0.05. DMSO was used at the same concentration of BAL27862.
Figure 6A. Representative image of CENP-B box FISH and mFISH stained RPE-1 metaphase spread cells. Chromosome 3 homologs are indicated by white arrows with single CENP-B box chromosomes signal magnified in insets. Scale bar represents 5 µm.B. Representative image of CENP-B box (red) and centromere 3 (green) FISH on RPE-1 interphase nucleus after 48 h IAA treatment. CENP-B box signal at individual homologs are magnified in insets. Scale bar represents 5 µm.C. Bar plots represent the quantification of the CENP-B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments. n = 69 cells. Unpaired t test, ****p < 0.0001.E. Immunoblot shows expression of the (ΔN)CENP-B-dCas9 after doxycycline induction.F. Representative images show a micronucleus or the main nucleus containing the Y chromosome (red) detected by FISH in no-doxycycline or doxycycline treated cells respectively. Chromosome X is shown in green. Scale bar represents 5 µm.G. Bar plot shows the mean (n ≥ 3) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM. n > 200 cells with a micronucleus. Mann-Whitney test. *p=0.0173.
Figure 7B. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent significant statistical difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. Im. St. = image stream.C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated conditionE. Representative image of a chromosome spreads in DLD-1 cells following mFISH and subsequently CENP-B boxes FISH. Inset shows the supernumerary (SN) chromosome. Scale bar represents 10 µm. F. Bar plot shows the mean variation in CENP-B boxes in DLD-1 cells from data from E (n = 10 cells) and whole-genome sequencing and centromere mapping ± SD. G-H. Scatter plot showing a significant positive correlation between the indicated features in DLD-1 cells (n = 1). The lines represent linear regression with 95% confidence band. Data from chr 13, 14, 21, 22, Y and SN were excluded from the analysis.J. Bars show frequency of micronuclei with or without siRNA treatment. Error bars represent the SEM of three independent experiments (n > 170 cells per replicate).K. (Left) Representative image of CENP-B box (red) staining after siRNA against CENP-A/HJURP for 48 h. CENP-B box signals at individual micronuclei are magnified in insets. Scale bar represent 5 µm. (Right) Bar graph represents the mean of the CENP-B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM. Color dots represent independent experiments. (n > 43 micronuclei and 840 main nucleus) Mann-Whitney U test: ***p = 0.0001.
Figure 8A-B. Scatter plot showing (A) a significant positive or (B) a negative correlation between chromosome length and the mis-segregation rate or CENP-C (n = 3) reads over chromosome length and mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. Numbers indicate chromosomes with low or high mis-segregation frequency.
Figure 1NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins.(B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins.(C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins.(D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins.(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.
Figure 2NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).(B) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(D) Body temperature of AdRiKO and control mice housed at 22°C (n=11 (control), n=9 (AdRiKO)).(E) Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n=8/group).(F) Body temperature loss upon cold exposure of AdRiKO and control mice (n=20 (control), n=17 (AdRiKO)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 3mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization. (A) Representative H&amp;amp;E staining of sWAT sections from AdRiKO and control mice (n=5/group).(B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group).(C) Glycerol in plasma of AdRiKO and control mice (n=6/group).(D) Representative H&amp;amp;E staining of BAT sections from AdRiKO and control mice (n=5/group).(E) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group).(F) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 4mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation. (A) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).(B) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(C) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group, each lane represents a mix of 3 mice).(D) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(E) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).(F) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=3/group).(G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).H) Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=9 (control 22°C), n=7 (AdRiKO 22°C), n=8 (control 4°C), n=8 (AdRiKO 4°C).(I) Respiration (VO2) of AdRiKO and control mice upon cold exposure (n=8/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 5mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). ((B) Extracellular acidification rate (ECAR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(D) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(E) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).(F) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)).(G) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 6Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group).(B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group).(C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group).(D) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII.(E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice).(F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group).(G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D.(H) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 1B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.
Figure 2B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.
Figure 3B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.
Figure 4B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.
Figure 5D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.
Figure 6D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.
Figure 7F. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs. The C666 cells were co-transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs for 24 h. The mRNA levels were determined using qPCR.G. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.H. Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1-specific siRNA or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV and analyzed by FACS at 24 hpi.
Figure 1B. Volcano plot of differently-regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation, with protein-level cutoffs set at log2 fold change >1.0 and -log10 adjusted P value >1.3 (P< 0.05), marked by dashed lines. Downregulated sites represented as blue points, and up-regulated sites as orange points (n = 2 independent experiments).C. Representative Western blots of the different MAPKs expressed in DRG neurons (DIV3) cultured in the presence or absence of NGF for 3h and subjected to a membrane/cytosolic fractionation. α-tubulin and STMN-1 are used as controls of cytosolic proteins and Na+/K+-ATPase and STMN-2 as controls of membrane proteins.D. Quantification of the ratio of protein localized in the membrane fraction after 3h of NGF deprivation (n = 6-14 biological replicates; series of unpaired t tests followed by a Holm-Sidak correction). Data information: Graphs represent mean ± SEM in (D) *p < 0.05 and **p < 0.01.F. Representative Western blots of total and palmitoylated DLK (upper panel) and quantification of DLK palmitoylation levels (lower panel) from cultured DRG neurons subjected to 3h of NGF deprivation (n = 5 independent experiments; Mann-Whitney U test). Data information: Graphs represent mean ± SEM in (F). *p < 0.05 and **p < 0.01.