EMBO/soda-nel huggingface dataset:
Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 (2021)
Figure 2

Figure 2. LMP1 localizes to the mitochondrial inner membrane and interacts with ANT1 in NPC cells.

(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).

(C) IP/IB analysis was used to detect the interaction of LMP1 with endogenous ANT1 in CNE1-LMP1-expressing cells.

(D) Detection of the LMP1-ANT1 interaction using a proximity ligation assay. Red fluorescence corresponds to the PLA positive signal and blue fluorescence corresponds to nuclei (DAPI staining; scale bar, 10 μm).

(E) LMP1 interaction with ANT1 in vitro. GST-ANT1 expressed in bacteria was incubated with a Flag-LMP1 protein and the proteins were subjected to Western blot analysis.

(F) ANT1 interaction with LMP1 is dependent on its domain 2 in 293T cells. ANT1 and LMP1 domain architecture and schematic representation of its respective deletion constructs used in this study in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 and Myc-tagged ANT1 mutants. IP of cell lysates using a Flag or Myc antibody.

(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.
Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 (2021)
Figure 3

Figure 3. EBV-LMP1 induces ANT1 conformational changes.

(A/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/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).

(E) Two tyrosine sites of ANT1, Y195/Y290 (human origin), were selected and mutated into TAG using a genetic codon extension technique to insert difluorotyrosine into their specific sites to construct two mutant prokaryotic expression vectors of ANT1Y195-F2Y/ANT1Y290-F2Y.

(F) Schematic flowchart of the incorporation of F2Y into ANT1.

(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.
Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 (2021)
Figure 4

Figure 4: EBV-LMP1 fixes ANT1 at the m-state to inhibit ADP/ATP exchange.

(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).
Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 (2021)
Figure 5

Figure 5. CATR enhances the sensitivity of NPC to cisplatin in vitro.

(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).

(B/C) Changes in mitochondrial membrane potential and viability of HK1-LMP1 cells were assessed 24 h after treatment with cisplatin (20 μM) alone or a cisplatin (20 μM) combination with BKA (1 μM)/CATR (1 μM). Mitochondrial membrane potential of cells was analyzed by using flow cytometry (B), and cell viability was measured by a CCK-8 assay (C). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).

(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).

(E) The combination index of cisplatin and CATR in HK1-LMP1 cells (note: CI < 1 indicates synergism, CI = 1 indicates an additive effect, and CI > 1 indicates antagonism).

(F) CCK-8 analysis of viability in HK1-LMP1 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 = 6, biological replicates per group, * P < 0.05, ** p < 0.01).

(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).
Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 (2021)
Figure 6

Figure 6. CATR enhances the sensitivity of EBV-LMP1-positive cells to cisplatin in vivo.

(A) The overall schematic diagram of the study design. The nasopharyngeal carcinoma xenograft model was established using HK1-LMP1 cells.

(B) Representative images of xenograft tumors from different treatment groups.

(C/D) The tumor volume (C) and tumor weight (D) of HK1-LMP1-derived xenograft tumors with various treatments. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).

(E) The mouse weight curve of the different treatment groups in a HK1-LMP1 cell xenograft model. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).

(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.
VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption. (2015)
Figure 1

VEGF-C is required for lymphatic vessel growth in the developing intestine and for the intestinal lymphatic vessel maintenance in adult mice

Mice received tamoxifen as indicated in the figure. Immunofluorescence analyses from the intestine were performed in (A–H) embryos, (I–L) pups, and (M–U) adults.

A–D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragment

E–H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.

K, L LYVE1 staining of intestinal lymphatic vessels at P6.

M–T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).

U Quantification of LYVE1 areas in (M–T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.

Data information: Scale bars: 200 μm in (A–H) and 400 μm in (K–T). n = 25 (M); 5 (N); 8 (O); 7 (P); 6 (Q); 5 (R); 6 (S); 3 (T).
VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption. (2015)
Figure 2

Smooth muscle cells are the main source of VEGF-C in adult intestine

Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B–F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.

Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZ mice.

Vegfc/LacZ β-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.

Vegfc/LacZ β-Gal staining and LYVE1 peroxidase staining.

Surface image of Vegfc/LacZ β-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).

Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).

Data information: Arrows indicate the VEGF-C expression in arterial SMC, arrowheads indicate the VEGF-C expression in SMC fibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.
VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption. (2015)
Figure 3

Intestinal lymphatic vessel regression leads to impaired lipid absorption and resistance to diet-induced obesity

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.

Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.

Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.

Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.

Total fat weight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.

Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.

Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.

Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wall LYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.

Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.

Data information: Scale bars: 100 μm (villi) and 300 μm (intestinal wall). Data are represented as mean ± SEM.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 1

Figure 1: Detection of pS65-Ub in vivo.

(A) Total Ub (left) and normalised pS65-Ub abundance (right) in 500 μg Ub-Clippase-treated, TUBE-enriched mitochondrial fractions from young (2-3 days) and aged (50-60 days) wild-type flies, an mtDNA mutator model (daG4>UAS-mito-APOBEC1), and wild-type flies that had been exposed to paraquat (5 mM) for 3 days.

(B) Absolute abundance of pS65-Ub in 4 mg mitochondria from young (2-3 days) wild-type flies following sodium carbonate extraction, protease treatment and phospho-peptide enrichment.

(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).

(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.

(E) Immunoblots for the indicated antibodies of (T) total lysates or (M) mitochondria-enriched fractions from fly heads (with proboscises removed) and bodies (thoraces and abdomens).

(F) Representative image of flight muscle from aged (50-day-old) wild-type animals immunostained with the indicated antibodies. Scale bars = 20 μm (inset, 5 μm).

Data information: Charts show mean +/- SEM, n = 3-4 independent biological replicates as shown.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 2

Figure 2: Analysis of the paraquat-induced mitochondrial ubiquitome from Pink1- and park-/- flies.

(A, B) (A) total Ub and (B) normalised pS65-Ub abundance in 500 μg TUBE-enriched, protease-treated mitochondrial fractions from wild-type, Pink1- and park-/- flies, either untreated young flies (2-3 days) or exposed to paraquat for 3 days.

(C) pS65-Ub immunoblot of mitochondria-enriched fractions following the paraquat pulse-chase assay in wild-type and park-/- flies. UT, untreated; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food. Asterisk (*) denotes non-specific band. See Figure EV1C for related control blots.

(D) pS65-Ub lane densitometry analysis of n = 3 independent replicates from (C), expressed relative to the most intense lane signal in each blot. Charts show mean +/- SEM.

(E-H) Relative abundance of (E) K6 chains, (F) K11 chains, (G) K48 chains, and (H) K63 chains in mitochondrial fractions treated as in A, normalised to the total mitochondrial Ub in each sample. Charts show mean +/- SEM from n = 3 independent biological replicates.

Data information: Statistical analysis used one-way ANOVA with Šidák's correction for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns = non-significant. The full list of multiplicity-adjusted P values is presented in Supplementary Table 1.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 3

Figure 3: Tissue level analysis of pS65-Ub accumulation in park-/- flies.

(A-B) pS65-Ub immunoblot of whole-animal lysates from untreated flies of the indicated genotypes. park RNAi was induced by da-GAL4 (ubiquitous), Mef2-GAL4 (muscle) or nSyb-GAL4 (neurons).

(C) pS65-Ub immunoblot of lysates from bodies or heads (without proboscises), as indicated, from park-/- animals.

(D) Flight muscles from approximately 3-day-old untreated park-/- flies immunostained for ATP5A and pS65-Ub. Scale bars = 10 μm.

(E) Higher magnification image of pS65-Ub positive structures in flight muscle counterstained for ATP5A. Scale bar = 5 μm. Chart shows quantification of ATP5A levels of pS65-Ub-positive (+ve) versus -negative (-ve) mitochondrial structures from 3 biological replicates (4-7 images per animal; average per animal shown in open circles). Line indicates mean. A.U. = arbitrary units. **** P < 0.0001, Mann-Whitney test.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 5

Figure 5: Loss of Atgs does not lead to the same degree of pS65-Ub accumulation as loss of parkin.

(A) pS65-Ub immunoblot of whole-animal lysates from wild-type, Atg5- and park-/- animals harvested at the indicated ages.

(B) pS65-Ub immunoblot of whole-animal lysates from young animals of the following genotypes: wild-type, Pink1-, park-/-, park RNAi (daG4>UAS-park RNAi), Atg1 RNAi (daG4>UAS-Atg1 RNAi), Atg8a-.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 6

Figure 6: pS65-Ub immunostaining of larval muscle.

(A-G) Representative images of pS65-Ub immunostaining false-coloured for intensity (Fire LUT) in muscle segments 6/7 from wandering L3 larvae of the following genotypes: (A) wild type, (B) Pink1-, (C) park-/-, (D) Atg5-, (E) Atg8a-, (F) Atg5-; park-/-, (G) Atg8a-; park-/-. Scale bar = 20 μm.

(H) Quantification of number of pS65-Ub puncta from A-G displaying mean +/- SEM from the indicated number of animals (n = 3-5 as indicated).

(I) Puncta volume from C-G expressed as mean +/- SEM (n = 4-5 animals).

Data information: Statistical analysis used one-way ANOVA with Dunnett's correction for multiple comparisons. Comparisons are against wild type unless indicated. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns = non-significant.
Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila (2022)
Figure 7

Figure 7: Parkin overexpression reduces pS65-Ub levels in an Atg5- background.

(A) pS65-Ub immunoblot of whole-animal lysates following paraquat pulse-chase assay of Atg5- flies with parkin overexpression (OE) (Atg55cc5 daG4>UAS-park) compared with Atg5- flies overexpressing mitochondrially targeted GFP (Atg55cc5 daG4>UAS-mito-HA-GFP). UT, untreated flies; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food.

(B) pS65-Ub lane densitometry, normalised to total protein (RevertTM stain), of n = 3 independent replicates of (A), expressed as pS65-Ub intensity relative to the most intense band in each blot. Charts show mean +/- SEM.

(C) Mass spectrometry analysis of normalised pS65-Ub levels in Ub-Clippase-treated, TUBE-enriched mitochondrial fractions from flies overexpressing parkin (daG4>UAS-park), Atg5- flies and Atg5- flies overexpressing parkin (Atg55cc5 daG4>UAS-park). Charts show mean +/- SEM (n = 3). Statistical analysis used One-way ANOVA with Dunnett's correction for multiple comparisons. ** P < 0.01; *** P < 0.001.

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CDK inhibitor Palbociclib targets STING to alleviate autoinflammation (2022)
Figure 2

Figure 2. Palbociclib is highly potent in abrogating STING activation.

(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.

(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.

(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.

(G-H) qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.

(I-J) qRT-PCR measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.

(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.

(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.