EMBO/SourceData huggingface dataset:
Figure 3 Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein.  Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.  HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot. ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3) ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3)  Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.
Figure 4 ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)  qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)  CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3)  The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)  Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm.  Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.  ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)  Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3)  ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)
Figure 5 CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h.  B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3)  The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)  Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm.  Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461.  G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)  Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.
Figure 6 qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)  pre-rRNA expression levels of PBMC from renal transplantation patients. (Stable group, n = 7. ABMR group, n = 8. TCMR group, n = 8).  Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5)  Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.  CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of skin-graft recipient mice were analyzed by FACS.  G-H ELISA analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (n = 5)  The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5)  Representative images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.  CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of heart-graft recipient mice were analyzed by FACS.  L-M ELISA analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)  qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)  FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.
Figure 2B: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.
Figure 3C: Biological consequences of the 2 identified N-terminal mutations. Both WT and mutant N-terminal protein fragments (p.Arg156Leu and p.Glu161Lys) were expressed in HEK293 cells as demonstrated by western blotting. The GFP-tagged protein products of both were not detected in the conditioned media of mammalian cells, as compared to wild type (WT), and appear to be retained in the intracellular compartment (mean ± s.e.m.; n = 3). Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media. UT - untransfected negative control; nd - not detected; *: p=0.01094 (WT vs p.Arg156Leu) and p=0.04408 (WT vs p.Glu161Lys) by 2-tailed t-test. It has to date not been possible to test cytoplasmic retention for the two C-terminal mutations due to difficulty expressing the full length or C-terminal protein fragment in mammalian cells.
Figure 4A-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).
Figure 5A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.B and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).D: Analysis of the migration index (see Methods).E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.F: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvaehead (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvaehead; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf - hours post fertilisation; dpf - days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.
Figure 1(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion).(E) ECG of patient 3 (subject IV:13) showing mild QTc prolongation.(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.
Figure 2(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)].(I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon](J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER.(K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.
Figure 3(A) Skin fibroblasts(left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs(center), which expressed the pluripotency markers NANOG and SSEA4(right). Scale bars: 100 μm.(B) TECRLHom-hiPSCs have a normal karyotype(left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right).(C) TECRLHom-hiPSCs generate derivatives of mesoderm(left), endoderm(center) and ectoderm(right). Scale bars: 25 μm(F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm.(G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs(left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs(right).
Figure 5(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2and fractional SR release in hiPSC-CMs.(C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.
Figure 1A, B Detection of γb palmitoylation through the biotin-switch assay in N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or γb3CS-3xFlag amounts recovered from the neutravidin beads. The loading controls indicate sample loading onto the neutravidin beads. Data information: In (A, representative data are shown and three biological replicates had similar results.D Western blot detection of viral accumulation in BSMV- and BSMV3CS-infected N. benthamiana plants with anti-TGB1 or anti-γb antibodies. SL8, eighth systemically infected leaf; SL9, ninth systemically infected leaf. Ino, agroinfiltrated leaves. Rubisco large subunit (RbcL) was used as a loading control. Data information: In representative data are shown and three biological replicates had similar results.F Western blot analysis of protein accumulation in BSMV- and BSMV3CS- systemically infected barley leaves with anti-TGB1 or anti-γb antibodies. RbcL was used as a loading control. Data information: In F), representative data are shown and three biological replicates had similar results.
Figure 2A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase (PEPC) was used to assess cytosolic contamination of isolated chloroplasts. Data information: , representative data are shown and three biological replicates had similar results.C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.
Figure 3A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm. B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas. Data information: In (A, representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody. Data information: In representative data are shown and three biological replicates had similar results.E Subcellular localization of BSMVγb-GFP and BSMV3CS-GFP in N. benthamiana leaf epidermal cells. The mCherry-HDEL and mTalin-RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above. Scale bars, 10 μm. Data information: In E), representative data are shown and three biological replicates had similar results.F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of γb with membranes was determined by 1% Triton X-100 extraction.
Figure 5B Analysis of γb palmitoylation in NbPAT15- or NbPAT21-silenced N. benthamiana plants. NbPAT15 and NbPAT21 were silenced by intron-spliced hairpin RNA-mediated RNAi approaches The γb-3xFlag plasmid was agroinfiltrated into the leaves, followed by biotin-switch assays at 3 dpi. Data information: In (B representative data are shown and three biological replicates had similar results.C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell-to-cell movement. Infiltrated leaves were observed by confocal microscopy at 3 dpi. Red fluorescence produced from the mCherry expression cassette shows the primary infection foci, and GFP fluorescence outside the red regions shows virus spread into secondary infected cells. Scale bars, 100 μm. Data information: In C), representative data are shown and three biological replicates had similar results.D Quantification of BSMV movement. The green and red fluorescent areas were measured by ImageJ software (n = 10). Data information: In (D), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).
Figure 6A, B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N. benthamiana at 3 dpi. PAT15-YFPn and PAT21-YFPn were co-expressed with BSMVγb-YFPc. Chloroplast autofluorescence is depicted as a false red color and mCherry-HDEL provides the ER marker. Figures on the right show the normalized fluorescence intensities of GFP, mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 20 μm. Data information: In (A, B, representative data are shown and three biological replicates had similar results.C Co-localization analyses of αa-GFP, RFP-TGB1, and CFP-γb in N. benthamiana leaves at 3 dpi. Mixtures of A. tumefaciens containing αa-GFP, RFP-TGB1, and CFP-γb were infiltrated into NbPATs-silenced N. benthamiana leaves or the non-silenced TRV:GUS control. Chloroplast autofluorescence is displayed as a false pink color. Figures on the right show the normalized fluorescence intensities of GFP, RFP, CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.D Co-IP assays of interactions between γb and TGB1 in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS and TRV:GUS combined with the 2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and IP proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. E Average amounts of RFP-TGB1 foci co-localizing with αa-GFP and CFP-γb at the chloroplast peripheries shown in panel C (n = 12). Data information: In E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).
Figure 7B Fractionation assay to detect γb membrane-associations. Separation of total protein (T) extracted from BSMV infected leaves into soluble (S) and membrane (M) fractions by ultracentrifugation at 100,000 g. PEPC and BiP, respectively, provide soluble and membrane fraction markers.C Aqueous two-phase partitioning of γb PM localization. The microsomal pellet from BSMVγb-GFP-infected leaves was obtained by ultracentrifugation at 100,000 g, and the two-phase fractionation procedure was used to separate the plasma membrane (upper phase) from other cellular membranes (lower phase). H+-ATPase is a PM marker.D PM associations of γb-GFP and γb3CS-GFP in N. benthamiana protoplasts. The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb-GFP and γb3CS-GFP. The third and fourth columns show γb localization in BSMVγb-GFP and BSMV3CS-GFP-infected leaves. PIP2A provided a PM marker (Nelson et al., 2007). Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In D, , representative data are shown and three biological replicates had similar results.E Western blot analysis of subcellular fractions of BSMV infected N. benthamiana leaves at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane associated γb was released by 1% Triton X-100 treatment.F Western blot detection of BSMV accumulation in N. benthamiana plants. A. tumefaciens mixtures harboring RNAα and RNAβmTGB2, along with RNAγ, RNAγγb-ROP, or RNAγγb-mROP, were agroinfiltrated into N. benthamiana leaves. Western blotting to detect virus accumulation at 3 dpi used anti-TGB1 or anti-CP antibodies. Actin provided loading controls.G Analysis of the effects of enhanced PM association of γb on cell-to-cell virus movement with the BSMV duplex fluorescence (dfBSMV) reporter system. Mixtures of A. tumefaciens containing dfBSMV, dfBSMVγb-GFP-ROP, and dfBSMVγb-GFP-mROP were infiltrated into N. benthamiana leaves. Infiltrated leaves were observed by confocal microscope at 3 dpi. Scale bars, 100 μm. (n = 6). Data information: In G), representative data are shown and three biological replicates had similar results. In (G), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).