EMBO/SourceData huggingface dataset:
Figure 5(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um.(B) The protein levels of IκBα and its phosphorylated form (p-IκBα) during mouse early embryonic development were detected by western blotting, representative western blotting of IκBα and p-IκBα was shown on the left and the quantitation analysis (right panel) indicated that the ratio of p-IκBα/total IκBα was gradually increased from MII oocytes to 4-cell stage embryos (three independent experiment replicates were performed, error bars represent s.e.m.).(C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test.(D, E) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA #1 or siRNA #2 or overexpression of Tab1 by Tab1 mRNA injection (shown in D. Scale bar = 25um.), the percentages of two-cell stage embryos with p65 nuclear translocation (shown in E.) were significantly decreased after Rnf114 knockdown or Tab1 overexpression (chi-square test was performed for statistical analysis).(F) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the Rnf114 siRNA #1, siRNA #2 injected groups.(G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.
Figure 1(A) Confocal observation of mitochondrial potential after TMRM staining of LMP1-negative or -positive NPC cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial potential. Images were analyzed using Image J software (scale bar, 10 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).(C) Extent of Ca2+-mediated mitochondrial swelling in NPC cells. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).
Figure 2(A) LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right).(B) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(G) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody.
Figure 3(A Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells.B) Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, ** p < 0.01).(C The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP.D) The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, * p < 0.05, ** p < 0.01).(G) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min. (H) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1-ΔT. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min.
Figure 4(A-B) The ADP/ATP exchange rate in EBV-LMP1-positive nasopharyngeal carcinoma cells is decreased. The fluorescence value of Magnesium Green ™ (1 μM) was measured at 505 nm (Ex)/535 nm (Em) by enzymography (A), and the ADP/ATP exchange rate was calculated after conversion to ATP (B). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, ** p < 0.01).(C) LMP1-negative or -positive NPC cells were treated with BKA (1 μM) or CATR (5 μM) for 24 h, Mitochondrial OCR assessed with the XF24 extracellular flux analyzer (Seahorse) under sequential treatment with oligomycin, FCCP, and antimycin A. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (D) Oxidative phosphorylation-related metrics based on OCR datas. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05,** p < 0.01).
Figure 5(A) HK1-LMP1 were treated with increasing concentrations of cisplatin for 24 h. Cell viability was determined by MTS assay. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(D) The IC50 value of the combination of CATR and cisplatin in CNE1-LMP1 cells was changed. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(G) Flow cytometry analysis of death of NPC cells treated for 24 h with CATR (1 μM) alone, cisplatin (20 μM) alone, or cisplatin (20 μM) combined with CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, * p < 0.05, ** p < 0.01).(H) Trypan blue staining was used to detect death of NPC cells treated for 24 h with vehicle, cisplatin, CATR, or a combination of cisplatin and CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).
Figure 6(F) Immunohistochemistry examination of the protein levels of Ki67 (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).(G) Apoptosis was analyzed by TUNEL assay (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (H) Apoptosis was analyzed by Western blotting.
Figure 4(A) Michaelis-Menten analysis of the reduction of 2-hydroxyacetophenone (2-HAP). The initial velocities (V0/[Etot]) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. The turnover rates (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. The data points show the average values for these technical replicates, while the error bars show the standard deviations.
Figure 6(A) Analytical gel-filtration of purified his8-MBP-SCR2 (black), strep-SCR (yellow), and his8-MBP-SCR2/strep-SCR on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The turnover rate (V0/[Etot]) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.
Figure 1Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs.Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, *P < 0.05, also hereafter in similar experiments). n = 3 biological replicates.  Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in (H) (left), and quantification of pluripotency proteins (right) (mean values ± SD). n = 3 biological replicates.Images of mESCs transduced with Rptor siRNA on different days in 2iL. siNC, transduced with a negative control; day 0 was the day of siRNA transfection, also hereafter in similar experiments. Scale bar, 400 µm.
Figure 2RNA-seq heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in 2iL.   RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL.
Figure 3Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (B) (mean values ± SD). n = 3 biological replicates.  Western blotting for the indicated shRNAs and pluripotency proteins as in (B) (left) and quantification (right) (mean values ± SD). n =3 biological replicates.
Figure 4Relative proliferation of mESCs as in (B) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, **P < 0.01, also hereafter in similar experiments). n = 3 biological replicates.Images of mESCs transduced with Larp1 shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.
Figure 5Relative enrichment heatmaps of mitochondrial ribosome subunit and ETC complex component mRNAs as in (A).RT-qPCR for pluripotency and mitochondrial ribosome subunit genes as in (E) (mean values ± SD). n = 3 biological replicates.
Figure 6OCR analysis of INK128 (200 nM) treated mESCs in 2iL.qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.Western blotting for the indicated pluripotency proteins in mESCs as in (F) (left), and quantification (right) (mean values ± SD). n = 3 biological replicates.
Figure 7Images of mESCs transduced with Rptor or Rictor shRNAs in SL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (A) (mean values ± SD). n = 3 biological replicates.  Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in SL. Scale bar, 400 µm.Western blotting for EIF4E, EIF4G1 and the indicated pluripotency proteins in cells with the indicated KD in the indicated culture conditions.
Figure 1Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4. Reaction products were separated on a 4-12% SDS-PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies.In vitro reconstitution of 60S ribosome UFMylation. Purified 60S ribosome was incubated with either UFL1 on its own or UFL1/UFBP1 complex together with UBA5, UFC1 and UFM1 for indicated time points at 30°C.Immunoblot showing formation of free UFM1 chains in the presence (Lane 3,4) and absence of UFL1/UFBP1 (Lane 1,2). (Bottom) Graphical representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis.Immunoblot analysing polyUFMylation (top) and UFL1 autoUFMylation (bottom) in the presence of indicated Lys to Arg (K-R) and Lys-less(K0) mutants of UFM1.Immunoblot showing in vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of WT, Lys-less (K0) and single lys mutants (K only) of UFM1.
Figure 2Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala (indicated as C-A) mutants of UFL1 to check for UFBP1 UFMylation (top), UFL1 autoUFMylation (middle) and substrate UFMylation (bottom).Single-turnover lysine discharge assays in the presence and absence of full length UFL1/UFBP1 complex. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. Top gel - LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained
Figure 3Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.Immunoblot showing in vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries.Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1/UFBP1 truncations as depicted in (E).
Figure 4In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies.Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.   Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates,  Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies.Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 (1 µM, 2 µM, 3 µM). UFL1/UFBP1 concentrations - 1 µM.
Figure 5Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1/UFBP1 complex.   In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1/UFBP1 complex.  Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1.
Figure 6In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1WT) and UFC1 lacking the N-terminal helix (UFC1ΔN).Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained (Representative of three independent experiments).H. Effect of deletion of N -term helix of UFC1 in 60S ribosome UFMylation. Purified 60S ribosomes (50 nM) were incubated with 0.5 µM UBA5, 1 µM UFM1, 100 nM UFL1/UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30°C. The reaction was stopped at indicated time points and run on a 4-12% SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. (Right - shorter time duration. Left - Longer time duration).
Figure 1B HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.C Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate nascent vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.
Figure 2A Quantification of the number of OCTμDsRed2μpositive/TOM20μnegative vesicles in cells expressing GFP, GFPμparkinWT, GFPμparkinR42P, GFPμparkinK211N, or GFPμparkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFPμparkin (gray bars) are indicated. Bars represent the mean ± s.e.m. Pμvalues are given first for GFPμ/GFPμparkinμpositive vesicles, then for total vesicle number (n = 49-68 cells in 2-3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).C HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.D Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 µM antimycin A (anti A, +) or DMSO (-) for 2 h.
Figure 3A Representative immunoblot of whole‐cell lysates from U2OS:GFP and U2OS:GFP‐parkin cells transfected with non‐targeting siRNA or siRNA targeting Drp1 (siDrp1).B U2OS:GFP−parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDHE2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm.C Quantification of PDH−positive/TOM20−negative vesicles in U2OS:GFP and U2OS:GFP−parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 48-85 cells in 2-3 experiments); ns, not significant; ***P < 0.001).D 5 μm‐long profile of the parkin‐positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A‐treated cell from (B) (left). Increasing position values on the x‐axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).E Quantification of the distance between the centres of GFP−parkin−negative (black bar) or −positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A−treated U2OS:GFP−parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t−test).F U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).G Quantification of TOM20−negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP−parkin (GFP−parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 24-59 cells in two experiments; ns, not significant; ***P < 0.001).
Figure 4AHeLa cells, transfected with siRNA targeting Drp1 and GFP‐parkin (green), were treated with 25 μM antimycin A for 90 min following a 30‐min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.B Quantification of PDH E2/E3bp‐positive/TOM20‐negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E‐64d; both total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P‐values are given for total vesicle number (n = 19 to 120 cells in 2-3 experiments; **P < 0.01; ***P < 0.001).CHeLa cells, transfected with GFP‐parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3bp bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.D Quantification of PDH E2/E3 bp‐positive/TOM20‐negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P‐values are given for the total vesicle number (n = 42-64 cells in 2-3 experiments); ns, not significant.
Figure 5A Representative immunoblot of whole‐cell lysates from HeLa cells transfected with non‐targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full‐length band.B HeLa cells transfected with GFP−parkin (green) and siRNA targeting PINK1 (siPINK1) or non−targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin−positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm.C Quantification of PDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).