EMBO/soda-nel huggingface dataset:
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 7

Figure 7. ENT3 supports virus uncoating and replication of SARS-CoV-2 requires functional ENT3.

(A) Illustration of strategy to track the viral uncoating upon infection.

(B-D) (B) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and subjected to live imagining 30, 60 and 90 minutes post-infection. The arrowheads indicated the Syto82+ virus puncta in the cell. Scale bars represent 10 µm. (C) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82+ cells (%) and (D) the mean fluorescent intensity (MFI) per Syto82+ cell (D). Results were generated from images of three biological replicates (n=3), each with 4 fields analyzed.

(E) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and co-stained with 100 nM Lysotracker Green, subjected to live imagining 60 minutes post-infection. Data were the representative images of two biological replicates (n=2). Scale bars represent 5 µm.

(F) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10) for 2 hours and then enriched the endosomes and lysosomes. EMCV particles in endosome- or lysosome-enriched fractions were quantified by plaque assay. Shown are combined results from three biological replicates (n=3).

(G-K) (G) Calu-3 cells were infected with SARS-CoV-2 original strain or delta variant at MOI=0.1 for 48 hours, harvested, and examined for SLC29A3 induction. (H) ENT3 knockdown in Calu-3 cells was performed by lentiviral shRNA system. The knockdown efficiency was confirmed by RT-qPCR. ENT3 knockdown Calu-3 cells were infected with SARS-CoV-2 delta variant and subjected for the viral replication analysis 24 hours post-infection. The virus released out of cells was measured by plaque assay (I), and the viral titer in the cells was measured by RT-qPCR (J). (K) The replication of SARS-CoV-2 delta variant in scramble and ENT3 knockdown Calu-3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 24 hours post-infection (MOI=0.1). The expression of EMCV-2A2B was determined by RT-qPCR. The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1. The SARS-CoV-2 E gene expression level was calculated relative to RPLP0 to normalize the cell number. Shown are combined results from three biological replicates (n=3).

Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.

Expanded View Figure legends
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 1

Figure 1. Mutant cybrids lacking CIV display different SC I+III2 species.

(A) Mitochondrial extracts from 143B cells, COX1∆ and COX2∆ mutant cybrids and their respective isogenic controls (CON-1 and CON-2) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The graphs (right) show densitometric quantifications of the signals normalized to CII subunit SDHA. The mean of three independent controls (CON) was set to 100 and all measurements were referenced to that value. The values represent mean ± SD (n=3-8). Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; β, p<0.01; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05; ∗∗p<0.01.

(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.

(C) BN-PAGE was followed by CI-IGA assay and immunoblotting with the indicated antibodies (in brackets). The identity of the MRC complexes and SCs is: I+III2+IV1, SC containing CI, CIII2, and CIV; I+III2, SC containing CI and CIII2; I+III2plus, SC containing CI, CIII2 and a COX1-submodule; III2+IV, SC containing CIII2 and CIV; I, complex I; III2, CIII dimer; IV2, CIV dimer; subCOX1, COX1-containing subcomplexes.

Data information: See also Figures EV1 and EV2.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 2

Figure 2. Complexome profiling reveals a differential reorganization of the MRC in CIV-KO cybrids.

(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses.

(B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.

Data information: See also Figure EV3.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 3

Figure 3. CIV assembly factors co-migrate with an atypical COX1-submodule that is present in control and COX2Δ cybrids.

(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa).

(B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.

Data information: See also Figure EV3.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 4

Figure 4. COX subunits physically bind SC I+III2plus.

A-C Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (A) CIV subunits COX1 and COX5B. (B) CI subunit NDUFB7. (C) CIII subunit CORE2. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.

(D) Structural arrangement of COX subunits detected in association with SC I+III2. Upper illustrations depict lateral views of human SC I+III2+IV1, where CI (aquamarine) and CIII2 (light green) are positioned along the inner membrane plane (dots). CIV appears as a smooth backbone with the associated COX subunits highlighted to show their positions relative to SC I+III2: COX7A2 (blue) and COX5B (red) attach COX1 (yellow) to the concave surface formed by CIII2 and the CI membrane arm, interaction further stabilized by COX7C (green) binding to CI. COX4 (magenta) interacts with COX1 on the distal side of CIV. Medium illustrations: matrix (M) view. Lower illustrations: intermembrane space (IMS) view. The figure was created with USCF Chimera v.1.12 using the 5XTH structure (Guo et al., 2017) from the PDB database.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 5

Figure 5. Assembly kinetics of CI and CIII subunits into the COX7A2L-containing SC I+III2.

(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1.

(B) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I+III2+IV1 from 143B cells and into SCs I+III2 from the CIV-KO cybrids.

(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies.

(D) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I+III2+IV1 from control cells and into SCs I+III2 from CIV-KO cybrids.

B-D Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.

A-C The identities of MRC complexes and SCs are as in Figure 1.

Data information: See also Figure EV4.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 6

Figure 6. Assembly kinetics of COX subunits into the COX7A2L-containing SC I+III2.

(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.

(B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012).

C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.

Data information: See also Figure EV4.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 7

Figure 7. Stabilized turnover rate of SC I+III2 in COX2∆ cybrids.

Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.

Data information: See also Figure EV5.
Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes (2020)
Figure 8

Figure 8. Multiple pathways for respirasomes stabilization.

(A) Respiratory chain enzyme activities of control (blue), COX1∆ (red) and COX2∆ (green) cybrids. Enzyme activities were measured in three experimental replicates. Data are expressed as the percentages of control cells, and indicated as means ± SD. Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05.

(B) Structural reorganization of SCs in the absence of CIV. In control cells (top), subunits COX4 and COX5A form an independent subcomplex that binds newly-synthesized COX1, allowing the completion of CIV assembly and its further integration into respirasomes through canonical pathways. In COX1∆ cybrids (middle), SC I+III2 is rapidly degraded due to the absence of stabilizing CIV components. In COX2∆ cybrids (bottom), SC I+III2 is stabilized by the binding of a 'non-canonical' stable COX1-submodule (comprising subunits COX1, COX5B, COX7A2 and the assembly factor HIGD2A), likely by keeping this SC in an assembly-competent state for the further incorporation of CIV subunits/modules up to completion of respirasome biogenesis.

EXPANDED VIEW FIGURE LEGENDS
Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival (2012)
f1

a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins. b, Total RNA was extracted from RagA+/+ (n = 3), RagAGTP/+ (n = 3) and RagAGTP/GTP (n = 2) MEFs and RagA mRNA expression determined by quantitative PCR (mean ± s.e.m.). c, Weights of RagA+/+ (n = 24), RagAGTP/+ (n = 52) and RagAGTP/GTP (n = 22) mice at birth (data are scatter dots, mean). d, Representative photographs of RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates. Scale bar, 1 cm. e, Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA+/+ (+/+), RagAGTP/+ (G/+) and RagAGTP/GTP (G/G) neonates immediately after Caesarean section (0 h) or after 1 h of fasting (1 h fast). f, Liver and heart extracts from RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins. g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P  0.05; **P  0.01; ***P  0.005.
Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival (2012)
f2

a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.). b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant. c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5). d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.). e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units). f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.). g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point. h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.05.
Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival (2012)
f3

a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 &mgr;m. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTP neonates. b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62. c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins. d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting. e, Recombinant LC3B–GFP was expressed in RagA+/+ and RagAGTP/GTP MEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B–GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTP MEFs. Scale bar, 10 &mgr;m. f, Localization of recombinant TFEB–GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.
Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival (2012)
f4

a, AMPK&agr;1/&agr;2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Whole-cell extracts were immunoblotted for the indicated proteins. b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins. c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005. d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 &mgr;m. e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins. f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting. g, Proposed model for constitutive RagA-induced neonatal lethality. Green and red boxes indicate active and inactive protein or process, respectively.
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 1

Figure 1. FUNDC1 is a novel MAM protein and accumulates in the MAM under hypoxic conditions

A. Immunoblots of subcellular fractions from HeLa cells exposed to normoxia. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MA: mitochondrial-associated membranes.

B. Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia (1% O2) for 5 h. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MA: mitochondrial-associated membranes.

C. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ** P < 0.01.

D. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Arrows indicate gold particles.

E. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-TOM20 antibody (12 nm gold particles). Bar = 500 nm.

F. Quantification of the MAM:mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic (1% O2) conditions; Data (mean ± s.e.m.) are from 3 independent experiments. n = 60 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria), ** P < 0.01.

G. Quantification of the MAM:mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic (1% O2) conditions. Data (mean ± s.e.m.) are from 3 independent experiments. n = 50 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria). NS means not significant.

H. The quantification of MAM counts from 50 cells, ***P < 0.001.

I. The quantification of FACL4 immunogold particles from 50 cells, *** P < 0.001.

J. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-FACL4 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC-gold particles and green arrows indicate FACL4-gold particles.

K. The ratio of FACL4 immunogold particles on the MAM and mitochondrion. n = 80 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria).

L. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-DRP1 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC-gold particles and green arrows indicate DRP1-gold particles.