EMBO/SourceData huggingface dataset:
Figure 4(F) Co-IP assays were performed in HEK293T cells to validate the association between 3×Flag-Rab11a, 3×Flag-Rab11b and endogenous KIF11. Lane 1: input lysate; lane 2: flow through after co-IP; lane 3-5: 3 washes; lane 6: on beads; and lane 7: immunoprecipitation elutes. Cells transfected with 3×Flag empty vector were uses as a negative control. Experiments repeated over 6 times.(G) Cells were transfected with mCherry-tagged WT, Rab11-S25N, and Rab11-S20V, fixed and stained for endogenous KIF11 (grey) and pericentrin (green). The mCherry signal (red) represents direct fluorescent signal visualized under confocal microscope. Arrows denote position of spindle poles defined by pericentrin. Images represent three independent experiments. Scale bars, 10 μ (H) Quantification of the percentage of bipolar, monopolar, or multipolar (3 or more) spindles observed in mitotic cells identified from each microscopic field. Data represents 10-20 independent fields per condition from 3 experiments.(I) Immunofluorescent staining for KIf11 (green) and E-Cad (grey) in WT and DKO mouse intestines. Yellow arrows point to Kif11+ spindles. Images represents at least 10 images per mouse and 5 mice for each genotype. Scale bars, 50 μm. (J) WT and DKO mice were injected with IdU 3 hrs before sacrifice to label new dividing cells. Immunofluorescent staining for KIf11 (green), E-Cad (grey), and IdU (red) were performed. Yellow arrows point to abnormal Kif11 localization in DKO cells. Scale bars, 50 μm.(L-M) Immunofluorescent staining for KIf11 (red) and spindle pole markers γ-tubulin or pericentrin (green) in WT and DKO mouse intestines. Images represent 3 mice each genotype. White arrows point to spindle pole. Scale bars, 20 μm, 10 μm.
Figure 5(B-C) HEK293T cells were transiently transfected with 3×Flag-Rab11a (or 3×Flag-Rab11b) and V5-tagged KIF11 truncates. Lysates were immunoprecipitated by anti-Flag antibody, and probed by an anti-V5 antibody to assess intracellular associations. Cells transfected by V5 empty vector were used as a negative control.(G) Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from cells treated by different inhibitors. DMSO data was quantified for 5 experimental replicates and all other treatments were quantified for 4 experimental replicates.(H) HEK293T cells were transiently transfected with 3×Flag-Rab11a, treated with nocodazole (100 ng/ml) or vehicle overnight, and lysates were used for Flag immunoprecipitation followed by immunoblot for KIF11. Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from control (n=6) and nocodazole-treated (n=9) cells.(L) KIF11 co-IP analysis were performed using WT and RAB11-KD CaCo2 cell lysates. The immunoprecipitates were probed for CLIP1, ZW10, CCAR1, USO1, and CCNB1. Experiments represent 2-4 replicates for each target.(N) Immunofluorescent staining for endogenous Clip1 (red) and Kif11 (green) in WT and Rab11 DKO mouse intestinal epithelial tissues. n=4-5 mice for each genotype. White arrows point to Kif11+ spindles. Scale bars, 20 μm.
Figure 6(B) Propidium iodide (PI) staining of WT, BKO, and DKO enteroids 3 days after 4-OHT treatment ex vivo. Note DKO enteroids had increased PI staining along the epithelial edge. Vehicle treated DKO enteroids were used as controls. Scale bars, 200 μm. (C) PI-stained areas were normalized to total enteroid area for each enteroid of different genotypes.Immunofluorescent staining for Kif11 (red) and E-cad (green) were performed on enteroids of various genotypes, all of which were treated with 4-OHT. Same experiments were done for DKO enteroids treated with vehicle or 4-OHT. White arrows point to Kif11+ spindles. Scale bars, 20 μm.(I-J) Numbers of pHH3+ or Lyz1+ cells were counted per enteroid of vehicle (n=11 for pHH3; n=8 for Lyz1) or 4-OHT treated DKO (n=9 for both pHH3 and Lyz1) enteroids from biological replicates.(K-L) Representative confocal fluorescent images of pHH3 (red) or Lyz1 (red) stained with E-Cad (green) from vehicle or 4-OHT treated DKO enteroids. Scale bars, 50 μm.
Figure 7(A) Immunohistochemistry for cleaved caspase 3 (CC3) was performed on TAM-treated enteroids of different genotypes. Black arrows point to CC3+ cells at the epithelial lining of DKO enteroids. Scale bars, 50 μm.Representative apoptotic genes, Anxa1, Anxa5, Bcl2l1 which were highly elevated in DKO intestines. n=3 mice for each genotype.(I) Immunofluorescent staining for CC3 (green) was performed on WT and Rab11 DKO intestinal epithelial tissues before and after tamoxifen treatment. Scale bars, 50 μm. (J) Quantification of CC3 positive cells per crypt-villus axis in WT and Rab11 DKO intestines. Results were obtained from 10-15 independent microscopic images taken from 4-5 mice for each genotype.(N) Immunohistochemistry for NFκB was performed on WT and Rab11 DKO intestinal tissues. (O) Quantification of ratio of nuclear NFκB+ cell number over total IECs per crypt-villus axis. Data were analyzed from 10-15 independent microscopic fields. n=4-5 mice for each genotype.(P) Immunofluorescent staining for p53 (green) and E-cad (red) in WT and Rab11 DKO intestinal tissues. Scale bars, 50 μm, 20 μm.
Figure 16mA/dA levels (mol/mol) of seven stages of Hydractinia development . purple area indicates the background level of 6mA contamination from the digestive enzymes used in the respective experiment. hpf: hours post fertilization. The number of biological replicates used for each sample are indicated by the amount of data points in the graph.Whole-mount immunofluorescence of 6mA from 16- and 64-cell stages of Hydractinia. Samples were RNase-treated. . In D, the scale bare is equal to 20 µm.
Figure 2Whole-mount immunofluorescence of anti-6mA in 64-128-cell embryos upon injection of shGFP (as control), shAlkbh1Relative quantification of anti-6mA signals from immunofluorescence images Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=135), shAlkbh1 (n=168), and rescue (n=140), where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.Quantification of shAlkbh1-electroporated embryos showing significantly higher level of 6mA/dA (P<0.05) compared to shGFP electroporated embryos and to wild type embryos at 64-128 cell stage.
Figure 3High EU incorporation in 64-cell but undetectable in 16-cell embryos of Hydractinia.RNase treatment abolishes the EU signal.
Figure 4Whole-mount image of EU incorporation signals at 64 cells upon injection with shGFP, shAlkbh1, rescue, and mutant-rescue solution Relative quantification of EU signals Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=86), shAlkbh1 (n=63), rescue (n=64), and mutant-rescue (n=66) where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.
Figure 5Rapid decline of m6A-marked maternal RNA occurs between the 2- to 16-cell stages, analyzed by UHPLC-QQQ of m6A/A (% in arbitrary unit) from four Hydractinia developmental stages.Replication stall at 8-16 nuclei following hydroxyurea treatment. The control shows a normal number of nuclei at the same developmental stage as treated one.Whole-mount immunofluorescence of anti-6mA in 32-cell embryos upon injection of ATP and m6ATP at 20 mM The uninjected control shows a normal level of anti-6mA at 32-cell embryos.
Figure 1Representative images and quantification of somal intensity per unit area of GFP-RBM3 i-neurons at 37°C or after 72h cooling at 32°C. Nuclei and cells are outlined by white and yellow dashed lines, respectively. N= 207 (37°C) and 220 (32°C) cells. Scale bar: 5 μm.Western blots and quantification of RBM3 and GFP normalised to GAPDH in GFP-RBM3 i-neurons at 37°C or 32°C (72h).
Figure 2(A-B) Median GFP intensity of BFP-positive GFP-RBM3 i-neurons measured by flow cytometry upon the sgRNA/Cas9-mediated KO of top 30 positive (A) or negative (B) regulator candidates. Statistical analysis is performed between the specific and non-targeting sgRNA groups for positive regulators (A) or between the specific sgRNA and reporter groups for negative regulators (B) within the 37°C or 32°C (72h) population.(C) qRT-PCR of RBM3 mRNA level normalised to 18s rRNA in i-neurons at 37°C or 32°C (72h).
Figure 3RT-PCR of RBM3 mRNA (Exon 2-4) in i-neurons at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are calculated based on the intensity of PE-included (red arrows) and PE-skipped (green arrow) isoforms.qRT-PCR using a combination of primers targeting Exon 3a, Exon 3 or Exon 4-5 quantifies the PSI values of RBM3 PE (Exon 3a, including both 3a-L and 3a-S) relative to Exon 3 or Exon 4-5 at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor.RT-PCR of RBM3 mRNA (Exon 2-4) in HeLa cells at 37°C and 32°C (48h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.Schematics and RT-PCR of RBM3 minigene (Exon 1-4), flanked by unique sequences (thick black bars) to distinguish it from endogenous transcripts during PCR amplification, expressed in HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.
Figure 4(A-B) qRT-PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA in WT i-neurons at 37°C (A) or 32°C (72h) (B), when NMD is blocked by SMG1 inhibitor. Statistical analysis is performed between the specific and non-targeting sgRNA groups. PSI values of RBM3 Exon 3a-L in control and HNRNPH1-knocked down K562 and HepG2 cells. RNA-Seq data from ENCODE Project, 2 isogenic replicates are included.(D-E) RT-PCR of endogenous RBM3 (D) and expressed RBM3 minigene (E) in control (scramble siRNA) or HNRNPH1-knocked down HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. The ratio of PSI values between HNRNPH1 KD and control is shown for only the SMG1 i-treated conditions.(F-G) RT-PCR of endogenous RBM3 (F) and expressed RBM3 minigene (G) in SMG1 inhibitor-treated control (untransfected) or FLAG-HNRNPH1-overexpressing (OX) HEK293T cells at 37°C. PSI values of RBM3 PE are shown in the graphs on the right respectively. WT i-neurons are transduced with lentiviral constructs expressing BFP (Control) or HNRNPH1-T2A-BFP (HNRNPH1 overexpression, OX) at 37°C and 32°C, followed by RT-PCR of endogenous RBM3 with 24h SMG1 inhibitor treatment. PSI values of RBM3 PE are shown in the graph on the right.
Figure 5Western blot and quantification of HNRNPH1 normalised to GAPDH in WT i-neurons at 37°C and 32°C (72h). RBM3 blots of the same samples are shown for comparison.RT-PCR of WT and delGGGG RBM3 minigenes in HeLa cells at 37°C or 32°C (48h) treated with SMG1 inhibitor. PSI values of RBM3 PE are shown in the graphs on the right.
Figure 1A. In the Discovery cohort, Kaplan-Meier estimates of disease-free survival (DFS) in years (y) in patients with low (red) versus high (blue) nuclear PRMT5 expression (upper panel) and in 2 groups of patients according to their treatment. Patients treated with tamoxifen (Tam, middle panel) and with aromatase inhibitors (AIs, lower panel). P-values are calculated with the logrank test. B. The same analyses were performed in the Validation cohort.
Figure 2A. GST and GST-PRMT5 fusion proteins were incubated with in vitro-translated ERα, the interaction was then visualized by Western blotting using an anti-ERα antibody. The corresponding Coomassie-stained gel is shown in the right panel.B. ERα/PRMT5 interaction was assessed by co-immunoprecipitation of MCF7 cell extracts treated or not with E2 for the indicated times, using an anti-PRMT5 antibody. The presence of PRMT5, ERα and β-actin was evaluated by Western blot analysis.C. The same experiment was performed for MCF7 cells treated or not with tamoxifen (Tam) for the indicated times. The scale bar is 10µM.D. Proximity Ligation Assay (PLA) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2, Tam 1 µM or both for 6 h. After fixation, PLA experiments were performed to evaluate the interactions between ERα/PRMT5 using specific antibodies. The detected dimers are represented by red dots. The nuclei were counterstained with mounting medium containing DAPI (blue) (Obj:X60). The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.F. Paraffin-embedded sections from a fresh tumor were incubated with or without G595, and PRMT5 and SDMA expression were assessed by IHC.
Figure 3C. MCF7 cells were treated with or without E2, Tam or both for 6 h. ERα methylation was determined by PLA using an anti-ERα and a pan methyl recognizing symmetrical dimethylation (SDMA). The scale bar is 10µM. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.D. The same experiment was performed with G595 to verify that ERα methylation was PRMT5-dependent. Red dots reflect ERα methylation. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. The sale bar is 40µM. Western blotting was performed to assess ERα, PRMT5 expression and the global profile of methylation using a pan SDMA antibody.E. MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti-ERα antibody. ERα, SDMA and GAPDH expression was evaluated in the input.
Figure 4B. MCF7 cells were treated with Tam for 6 h in the presence or absence of G595, cell extracts were then subjected to ChIP assay using anti-ERα or anti-SMRT antibodies. The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters. The results are expressed relative to the signal obtained from input chromatin. The mean ± SEM of at least three experiments is shown. P-values were determined using a Student t-test.C. MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT-QPCR with specific primers for GREB1. The values were normalized against 28S mRNA and represent the ± SEM of three experiments.
Figure 5B. MCF7 cells were treated with Tam for 6h in the presence or in absence of G595, then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins. The scale bar is 10µM. Quantification was performed as in Figure 2. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. Western blotting was performed to assess ERα, SIRT2, SAP18 and GAPDH expression and the global profile of SDMA.
Figure 6A. ERα methylation SDMA was studied by PLA, and PRMT5 and MEP50 expression were analyzed by IHC on formalin-fixed MCF7 xenografts treated or not with Tam (Obj: X40). The scale bar is 50 µM. Histogram quantifying the number of dots/cells and the H-scores for nuclear PRMT5 and MEP50 are presented on the right. The mean ± SEM of 7 tumors is shown. P-values were determined using a Student t-test.B. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed PDX model HBCx-34 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 3 tumors is shown. P-values were determined using a Student t-test.C. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed the PDX model HBCx-22 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 7 tumors is shown.
Figure 7A. For each tumor, we analyzed the level of ERα SDMA methylation by PLA. The H-score for nuclear PRMT5 expression and the number of dots/cell are listed under the figures. The scale bar is 20µM.
Figure 1A. Volcano plot depicting differential expression of nearly 18000 unique genes (minimum expression of 1 read per kilobase per million, RPKM) in response to AMV infection. In red, upregulated genes (log2FC ≥ 1; adjusted p-value ≤ 0.05); in blue, downregulated genes (log2FC ≤ -1; adjusted p-value ≤ 0.05D. RT-qPCR analysis of expression of four upregulated m6A pathway-related genes in response to AMV infection (three biological replicates). Error bars represent the standard error of the mean (SEM). Asterisks indicate a p < 0.05 applying Student's t-test for ΔCt mean values (n = 3).
Figure 2A-D. RNA blots were hybridized to AMV-specific probes to reveal accumulation of viral RNA in double (de) and triple (te) ect2/3/4 (A, B) or ect2/3/5 (C, D) mutants compared to WT plants. A and C show local infection at 3 days post-inoculation (dpi); B and D show systemic infection in aerial tissue at 7 dpi. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Dashed lines indicate non-contiguous samples analyzed on the same membrane. Ethidium bromide staining of ribosomal RNAs (EB) was used as RNA loading control. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (*: p < 0.05; **; p < 0.01) using the Student's t-test (n = 4). AU, arbitrary units.
Figure 3A, RNA-blot analysis of AMV systemic infection in plants expressing ECT2WT-mCherry (WT) or ECT2W464A-mCherry (W464A) in the de23 background (A) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In A, upper and lower panels show independent complementation transgenic lines, and different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).B. RNA-blot analysis of AMV systemic infection in plants expressing in vir-1 plants (B) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In B, dashed line indicates non-contiguous samples that are analyzed on the same membrane. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (**: p < 0.01) applying Student's t-test (n = 4). AU, arbitrary units.
Figure 4A. ADAR expression (TPM - Transcripts Per Kilobase Million - mapping) in five samples of ECT2-FLAG-ADAR (S1-S5) and FLAG-ADAR (S6-S10) transgenic lines.B. Percentage of total reads mapping to the AMV RNAs in the different samples.
Figure 5A, Systemic infection of rosettes at 7 days post-inoculation (dpi) (A) Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).B. Systemic infection of rosettes and floral stems at 20 dpi (B). Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).
Figure 6A. Schematic representations of the annotated ECT2 gene (AT3G13460.1, top) or protein (bottom) showing the deletion of the Tyr-rich region (ΔN5) in ECT2ΔN5-mCherry lines.C. Protein blot developed with mCherry antisera to show accumulation of ECT2WT-mCherry or ECT2ΔN5-mCherry fusion proteins in pools of the same samples used in panel B. Proteins on the membrane were stained with Coomassie-blue as loading control (lower panel).E. Phenotypes of seedlings and young rosettes of the indicated genotypes at 9 and 16 days after germination (DAG). Scale bars: black, 1 mm; white, 1 cm.
Figure 1A HeLa cells were transfected with control (Ctr) or three different GCN2-targeting siRNA-s and progression from prophase to anaphase was observed by live-cell imaging. Averages (lines) and SEM (bands) from five (siCtr) or four (each siGCN2) independent experiments are shown; n= 379 for siCtr, 241 for siGCN2 #1, 170 for siGCN2 #2, 239 for siGCN2 #3. ****p<0.0001 for each of the siRNA-s (Kolmogorov-Smirnov test). t=0 denotes entry into prophase as judged by the first frame showing chromatin condensation.E, Chromosome congression is affected by the absence of GCN2. E Immunofluorescence images of HeLa cells stained for a kinetochore marker, CREST (grey) and DAPI for DNA (blue). Arrowheads indicate misaligned chromosomes. Scale bars represent 5 µm.G­­ Increased number of mitotic cells with tripolar spindles after GCN2 knock-down. HeLa cells were transfected with control (Ctr) or GCN2-targeting siRNA and fixed for immunofluorescence. The number of spindle poles was determined by using a pericentrin antibody. Mean and SEM are shown from five independent experiments. n= 61, 27, 19, 24 and 28 for siCtr; n=49, 29, 22, 26 and 26 for siGCN2; p=0.0125, paired two-tailed t-test.
Figure 2A, B There is no significant difference in global translation rates in mitosis after GCN2 knock-down. A HeLa cells were pulse labeled with OPP and fixed with 70% ethanol. OPP was detected by Click-IT (AF 647) and mitotic cells were identified by staining for phospho-histone H3. 2D flow cytometry plots are shown, where AF647 intensity corresponds to OPP incorporation. Translation levels normalized to siCtr G1. The graph shows averages and SEM from three independent experiments. Paired two-tailed t-tests.D, Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes (D) Representative immunoblots of three biological replicates are shown.E Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes Representative (E) quantifications of three biological replicates; averages and SEM are shown. Values are normalized to t=0.
Figure 3A Verification of the mutants. HeLa cells transduced with the indicated GCN2-siR constructs were transfected with control (Ctr) or GCN2-targeting siRNA. Halofuginone (HFG) was added to 40 nM for 4 hours, and GCN2 autophosphorylation, ATF4 induction and eIF2α phosphorylation were assessed by immunoblotting. γ-tubulin is shown as loading control. The line in the GCN2 blot indicates that a longer exposure of the same blot is shown on the left than on the right.C, GCN2 kinase activity is regulated in the cell cycle. HeLa cells were arrested at the G1/S transition using thymidine and released from the cell cycle at time 0 h. Samples were taken at the indicated time-points for immunoblotting Aurora A and Aurora A-P are shown to indicate the timing of mitosis, GCN2-P is shown to assess GCN2 activity. Representative immunoblot of GCN2 phosphorylation normalized to GCN2 (total), three biological repeats, three technical repeats each.