EMBO/SourceData huggingface dataset:
Figure 4Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.
Figure 5m6A levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to LC-MS/MS analysis to quantify m6A levels (presented as the m6A/A ratio). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.
Figure 6MeRIP (m6A-IP)-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls. : In (B) , data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.The expression of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14 KO, KO+WT, and KO+RK mESCs were subjected to Western blot analysis using the indicated antibodies.
Figure 7mESCs expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured.   Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.   In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05.
Figure 1Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. Images are shown in (A); scale bars = 5 mm.Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (each plant) (C) of the indicated genotypes were measured. SDs (n > 8 independent seedlings) are indicated.Distribution of lateral root primordia stages. Seedlings of the indicated genotypes were grown in 1/2 MS treatment with or without UV-B (1 W/m2) for 2 weeks. SDs (n > 8) are indicated.GUS staining of seedlings expressing DR5p::GUS transgene in the WT, uvr8 and rup1 rup2 background. Seedlings were grown in 1/2 MS with or without UV-B for 2 weeks. Images of lateral root primordia (E) and lateral roots (F) are shown. Scale bars = 100 and 50 μm, respectively.
Figure 2Phenotypic analysis. Seedlings of the indicated genotypes were grown in 1/2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV-B (1 W/m2) light for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (A) and average length of lateral roots (B) of the indicated genotypes were measured.UV-B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8-dependent manner, since the response to NAA was not repressed in uvr8. Seedlings of the indicated genotypes were grown in LD (16-h light/ 8-h dark) conditions for 5 days, then transplanted to new plates containing 0.4 μM NAA and kept in continuous white light or white light plus UV-B (1 W/m2) for 7 days. Images are shown in (C); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (D) and average length of lateral roots (E) of the indicated genotypes were measured.
Figure 3Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated.UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated.
Figure 4Transcriptome analysis of gene expression regulated by auxin, UV-B, and UVR8. (A) Heat map of UV-B-, UVR8-, and auxin-regulated genes. The parameter measured by color key shows the Log-fold change. (B) Auxin signaling genes are up-regulated by auxin treatment but down-regulated by UV-B in a UVR8-dependent manner. Three biological replicates were analyzed and final Log fold-change is shown.Quantitative RT-PCR results showing that UV-B represses the expression of auxin signaling genes. Six-day-old WT seedlings grown in continuous white light were transferred to UV-B light (2 W/m2) for the indicated period. Error bars are SD of three biological replicates.Quantitative RT-PCR analysis of auxin signaling gene expression in wild-type (Col-0), uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 5 days were kept in white light (D) or transferred to white light plus UV-B (E) for 1 day with the addition of 1 μM IAA. Error bars are SD of three biological replicates.qPCR analysis of auxin signaling gene expression in roots of WT, uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 12 days were kept in white light (F) or transferred to white light plus UV-B (G) for 1 day with the addition of 0.4 μM NAA. Error bars are SD of three biological replicates.
Figure 5In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB73 and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB77 (B) and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.BiFC assay showing that UVR8 interacts with MYB73/MYB77 in white light condition. N. benthamiana was co-transformed with MYB73-nYFP/MYB77-nYFP and cCFP, nYFP, and UVR8-cCFP, or MYB73-nYFP/MYB77-nYFP and UVR8-cCFP. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bar = 20 μm.Co-IP assays showing that MYB73 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8, MYB73-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.Co-IP assays showing that MYB77 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8 , and MYB77-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.
Figure 6Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated.Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.
Figure 7Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor.EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C)EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D).ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).
Figure 2Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.
Figure 3Co-immunoprecipitation of TgCRMPa and TgCRMPb. Lysates from parasites co-expressing TgCRMPa-FLAG3 and TgCRMPb-HA3 were split and incubated with either anti-FLAG (left panels) or anti-HA beads (right panels). Eluates were subjected to SDS-PAGE and immunoblotted with anti-HA and anti-FLAG Abs. Untagged, TgCRMPa-FLAG3 and TgCRMPb-HA3 parasites were used as controls. TgCRMPs robustly associate with each other.Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.
Figure 5Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.   Quantification of the dot pattern shown in (C). The values are reported as in (B).  Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.   Quantification of the dot pattern upon natural (+H F Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).
Figure 6Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed. Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.
Figure 1(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *.(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.(D) WB of H1299 cells transiently expressing the indicated constructs.(E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53.(F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB.(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.
Figure 2(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.(B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).
Figure 3(A) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm.(B) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications.(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.
Figure 4(A) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53's translation initiation (MO)(B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53's translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.(C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53's translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data.(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.(F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53's translation initiation site (MO) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.
Figure 2A. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.