EMBO/soda-nel huggingface dataset:
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 7

Fig 7. In vivo edaravone treatment rewires the ROS-driven lipogenic switch and prevents IR. Data on wild type (wt, black bars and traces) and LowOXPHOS (orange bars and traces) mice treated or not with 3 mg/kg edaravone are shown.

A. Mitochondrial ROS after 3 hours treatment with 2 μM edaravone (green bars), 10 nM MitoQ (purple bars) or 1 mM NAC (blue bars). Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition.

B. Representative WB expression of Skm ROS system proteins in mice administered (30 days) edaravone. Two samples per condition are shown. Each sample contains extracts from 3 mice (wt + edaravone, n= 6; LowOXPHOS + edaravone n= 6). Peroxiredoxin 2 and 3 (PRX), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. GAPDH is presented as a loading control.

C. qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone (green bars), 10nM MitoQ (purple bars) or 1 mM NAC (blue bars). 6 replicas/condition.

(D, E) Representative WB expression of Skm de novo lipid synthesis (D) and OXPHOS (E) proteins. One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice (D) and rewired the upregulation of ETF-A, ETF-B and SDHA proteins to the levels of wt (E). Two samples per condition are shown. Each sample contains extracts from 3 mice. Tubulin or βF1 is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).

F. 2D-PAGE of Skm extracts from wt (n= 3) and LoxOXPHOS (n= 3) mice treated with edaravone. The pI of SDHA, calculated by protein migration in pH 3-10 NL strips, was the same for both genotypes.

G. Representative blue native immunoblots (BN) of Skm mitochondrial membrane proteins from 2-month-old mice treated with edaravone for 30 days (wt + edaravone, n= 3; LowOXPHOS + edaravone, n= 3). The migration of the respiratory complexes/supercomplexes CI-CIV and hATPIF1 is indicated. VDAC is shown as a loading control.

H. Mouse body weight following the administration of HFD and edaravone.

I. v-WAT amounts at day 60th of HFD in mice treated with edaravone.

J. Insulin (ITT) and glucose (GTT) tolerance tests after 60 days of HFD in mice treated with edaravone.

K. GTT after 80 days of HFD (left) and ITT after 90 days of HFD (right) in wt and LowOXPHOS mice treated with edaravone, showing that the compound has an improving effect itself.

L. Blood glucose following the administration of HFD. T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone.

Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test. See also Fig EV5.
ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase (2007)
Figure 1

Involvement of Drosophila Autophagy‐specific gene 1 in Drosophila Target of rapamycin‐dependent cell growth and development. (A) Genomic structure of CG10967. The P‐element insertion site of EP3348 (DmATG11) is denoted. (B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT–PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d. (C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg‐laying (AEL), and quantification of the larvae that developed into the mid–late third instar stage of each genotype (bottom); n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment. (D) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1. The fat body images of the second/early third instar larvae of denoted genotypes under fully fed conditions. (E) Images of the larval salivary glands of denoted genotypes. Hoechst33342 (pseudo‐coloured green) and phalloidin‐TRITC (red) were used to visualize the nuclei and cell boundary of larval salivary gland cells, respectively. (F) Quantitative analysis of cell and nuclear sizes in Fig 1E; n=5. Bars indicate mean±s.d. DmATG1, Drosophila Autophagy‐specific gene 1; dTOR, Drosophila TOR; qRT–PCR, quantitative real‐time reverse transcriptase–PCR.
ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase (2007)
Figure 2

Functional interaction between Drosophila Autophagy‐specific gene 1 and Drosophila S6 kinase. (A) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment. (B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulin immunoblot was used as a loading control. Rheb expressing flies (hs>Rheb) were used as a positive control (C). hs>Rheb larvae were subjected to heat shock at 37°C for 1 h and then incubated at 30°C for 3 h before sample preparation to induce Rheb expression. For quantification, the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop, and normalized to the tubulin levels. Results are expressed as a fold change compared with the wild‐type controls (w1118). DmATG1, Drosophila Autophagy‐specific gene 1; dS6k, Drosophila S6 kinase.
ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase (2007)
Figure 3

Inhibition of S6 kinase phosphorylation by Autophagy‐specific gene 1. (A,B) HEK 293T cells transfected with denoted plasmids were (A) deprived of nutrients for 90 min, and then replenished with DMEM for 30 min or (B) stimulated with 50 ng/ml epidermal growth factor (EGF) for 30 min. The S6K immune complexes were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel). (C) HEK 293T cells transfected with control siRNA, ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min, and then the phosphorylation and protein levels of endogenous S6K (top two panels) and S6 (bottom two panels) were determined. (D) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The phosphorylation and protein levels of endogenous S6K were examined. For quantification, the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop, and normalized to the S6K protein levels. Results are expressed as a fold change compared with the first indicated sample. (E) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP‐C1 (a transfection marker, green), and nutrient‐starved for 90 min, fixed and stained with phosphospecific S6 antibody (red). ATG1, Autophagy‐specific gene 1; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; siRNA, short interfering RNA; S6K, S6 kinase; WT, wild type.
ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase (2007)
Figure 4

Inhibition of S6 kinase Thr 389 phosphorylation by Autophagy‐specific gene 1α. (A,B) HEK 293T cells were transfected with HA–Akt (A) or Myc–RSK (B), and/or Flag–ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel). (C) HEK 293T cells were transfected with HA–S6K Thr 389 Glu mutant and/or Flag–ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti‐phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy‐specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; RSK, ribosomal S6 kinase; WT, wild type.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 2

Figure 2. Ultrastructure of PC-free yeast cells

A-L Wild type cultured in C- (A), cho2opi3 cultured in C+ (B ), coS#4 (C-D), coS#3 (E-G), and coS#2 (I-K) cultured in C- were analyzed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L).
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 4

Figure 4. The lipidome of PC-free cho2opi3 suppressors shows increased lipid content and shortening of average acyl chain length

Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition.

Membrane lipid class composition of classes contributing at least 1% of total membrane lipids, the inset shows CDP-DAG and the separate lyso(L)-phospholipids (lyso-PL);

Fatty acyl chain profiles of the total lipid, the membrane glycerolipid (ML) and the TAG fraction showing acyl chains that contribute at least 1% of total, with the C10-C14 acyl chains in the insets;

PE molecular species profile (sum of carbon atoms in the acyl chains : sum of double bonds in the acyl chains) showing species that contribute at least 2% of total PE;

(E-F) Percentage of molecular species containing more than 32 carbon atoms in both acyl chains (C34+C36) (E) and of saturated acyl chains (SFA) (F) in the membrane glycerolipids (ML) and the major membrane lipids; of the indicated strains cultured to mid-log phase in SD C+/-.

Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 5

Figure 5. The PDAT Lro1 is essential in cho2opi3 suppressors

Scheme showing the reaction catalyzed by Lro1.

Generation of suppressors of choline auxotrophy of cho2opi3, cho2opi3lro1, and cho2opi3lro1 pLRO1 on choline-free medium as indicated at 30 oC for 7 d.

Three suppressor clones derived of cho2opi3 and cho2opi3lro1 transformed with plasmid pLRO1 were spotted on SGal ura- plates without choline, incubated at 30 oC for 4 d, replica-plated onto SGal C+ and C- plates containing 5-FOA and uracil, and incubated at 30 oC for 7 d.

Serial dilutions of the strains indicated were spotted on SD C+/- and incubated at 30 oC for 6 d.

Growth (30 oC for 6 d) and phospholipid composition of co acc1N/H lro1 pLRO1 cultured in SD C- ura-, containing glucose/galactose mixtures (2%, w/v) as carbon source with the percentage of galactose indicated. Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 6

Figure 6. Inhibition of Acc1 activity by soraphen A suppresses choline auxotrophy of the cho2opi3 parent by reducing average acyl chain length, and abrogates growth of cho2opi3 suppressors

Ten-fold serial dilutions of 1 OD600 unit/mL of the strains indicated were spotted on SD C+/- containing 0.05 or 0.25 μg/mL SorA, and incubated at 30 oC for 3 d. Control plates contain 0.02% (v/v) ethanol.

Lipid-incorporation of [1-14C]acetate after a 1 h pulse in coS#2 and co acc1N/H relative to the cho2opi3 parent strain during culture on SD C+ medium. Data are shown as mean of 2 biological replicates.

ACC1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.

(D-E) Fatty acyl chain profile of the total lipid fraction (C) and PE molecular species profile showing species that contribute at least 1% of total PE (D) of cho2opi3 transferred to SD C+ and SD C- containing 0.05 μg/mL SorA as indicated, and cultured from starting OD600 0.02 to mid-log phase. Data obtained by mass spectrometry are presented as mean ±SD (n=3 biological replicates).

(F-G) EM analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to mid-log phase in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 7

Figure 7. mRNA profiling of evolved cho2opi3 2n-1 suppressors reveals upregulation of ORE genes, however β oxidation is not required for suppression of choline auxotrophy

Heatmap showing log2 fold changes of mRNA expression in the cho2opi3 parent strain cultured in SD C-I- for 13 h vs. wild type, and in co S#3, #4, and #5 cultured to mid-log phase in C-I- vs. wild type and vs. parent. Transcripts changing more than 1.7-fold (p < 0.01) in at least one of the comparisons are depicted. Hierarchical clustering was by average linkage (cosine correlation); (functional) categories of enriched transcripts were assigned per cluster.

Transcript profiles of the PC-depleted parent and evolved suppressors vs. wild type of UPR-induced genes (Kimata et al., 2006; Travers et al., 2000) changing more than 1.7 fold (p < 0.01) in the parent, and of ORE-genes from the cluster in Fig. 6A. Heatmaps show log2 fold changes according to the color scales; asterisk (*) indicates gene on chromosome XV.

POX1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 8

Figure 8. Suppression in 2n-1 suppressors depends on inhibition of Acc1 by an increased concentration of acyl-CoA

Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.
Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast (2021)
Figure 9

Figure 9. Membrane lipid packing in yeast cells with and without PC

Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.

Comparison of average GP-values ±SD from at least 7 different images per strain and condition from two independent experiments, with the datapoints representing the average GP-values of the separate images. Statistical comparison by GraphPad Prism one-way ANOVA: **p<0.01, ****p<0.0001.
Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1‐mediated nuclear export (2008)
Figure 1

Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR‐ and NucNeoR‐expressing cells. RCC1.24 cells were transfected with the NeoR (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c‐myc antibody 9E10. While NeoR was evenly distributed in nucleus and cytoplasm (a), NucNeoR was confined to the cell nucleus (b). Daylight photographs of the stained cells are shown in (a’) and (b’). (C) NeoR‐ and NucNeoR‐transfected RCC1.24 cells were treated with 100 U/mL IFN‐γ for 48 h to induce MHC class II expression. Subsequently, the cells were washed and co‐cultured with the CD4+ T cell clone 20‐4/A4, which recognizes a peptide derived from NeoR on HLA‐DP3. GM‐CSF secretion by the T cells was measured 20 h later by ELISA. Both transfectants were recognized after IFN‐γ treatment, indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation. (D) LCL1.11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR‐specific T cell clone 20‐4/A4. Antigen‐transfected LCL1.11 cells were recognized irrespectively of NeoR localization, while untransfected cells or cells transfected with a GFP‐expressing control plasmid were not recognized.

WILEY‐VCH
Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1‐mediated nuclear export (2008)
Figure 2

MHC class II‐restricted presentation of NeoR and NucNeoR is blocked by inhibition of autophagy and lysosomal degradation. (A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing. (B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing. (C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin. (D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold. (E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.

WILEY‐VCH
The Mutational Landscape of the SCAN-B Real-World Primary Breast Cancer Transcriptome (2020)
Figure 2

Fig 2: Overview of non-synonymous mutations in terms of base substitution signatures, molecular subtype, and protein impact

A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.

B) Number of non-synonymous mutations per sample. Bars are colored by PAM50 subtypes Luminal A (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray).

C-F) Lollipop plots showing the location, abundance, and impact of SNVs in C) TP53, D) PIK3CA, E) PTEN, and F) ERBB2 on the respective encoded protein. Protein change labels are shown for the most mutated amino acid positions, with residues ordered left to right by mutation frequency within each label.
The Mutational Landscape of the SCAN-B Real-World Primary Breast Cancer Transcriptome (2020)
Figure 5

Fig 5: Impact of gene mutations on overall survival across treatment groups

A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.
The Mutational Landscape of the SCAN-B Real-World Primary Breast Cancer Transcriptome (2020)
Figure 6

Fig 6: Impact of pathway mutations on overall survival across treatment groups

A-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV4. In each Kaplan-Meier plot, wildtype (wt) cases are plotted in blue, mutated (mut) cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. See Table EV3 for Reactome pathway IDs. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.