EMBO/soda-nel huggingface dataset:
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 7

Figure 7. TMEM33 expression is positively correlated with plasma cholesterol levels in mice.

(A) Representative images from the Adeno vector control and TMEM33 expressing groups. Scale bar, 1.5 cm.

(B) Time course of bioluminescent intensity of the mice injected with a control adenovirus and TMEM33 expressing adenovirus (n=7).

(C) The total serum cholesterol levels in mice before and after control adenovirus and TMEM33 expressing adenovirus injection on days -1 and 5 (n=7).

(D) Genotyping of CRISPR/Cas9 generated TMEM33 knockout mice using fluorescent probes for PCR. FAM+, Cy5- indicates the TMEM33+/+ genotype; FAM+, Cy5+ indicates the TMEM33+/- genotype; and FAM-, Cy5+ indicates the TMEM33-/- genotype (n>=5).

(E) Representative H&E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers. Scale bars, 100µm.

(F, G) Significant decrease of total plasma cholesterol (F) and LDL-C (G) levels in TMEM33-/- and +/- mice relative to WT counterparts (n>=5).

Data information: Data are displayed (B, C, F and G) as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 8

Figure 8. Systemic inhibition of PKM2 in vivo increases serum cholesterol levels and promotes allografted tumor growth.

(A) Systemic PKM2KO results in increased total plasma cholesterol and LDL-C levels in mice (n=8).

(B) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice (n=5).

(C) Quantification of the averaged tumor weights in (B) (n=5).

(D) TEPP-46 treatment increased total plasma cholesterol and LDL-C levels in mice (n=8).

(E) The growth curves of py8119 allografts treated with vehicle and TEPP-46 (50mg/kg) for 17 days in mice (n=7).

(F) Tumor weights of PKM2 WT py8119 allografts treated with vehicle or TEPP-46 (n=7).

Data information: Data are displayed (A-F) as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Source data are available online for this figure.

Expanded View (EV) legends
Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ (2013)
f1

a, Fluorescence microscopy HeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by &agr;-GM130 antibodies. Scale bar, 10 µm. b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm. c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm. d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments. e, Number of recoverable c.f.u. per mouse lung 24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P  0.001.
Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ (2013)
f2

a, Sequence–structure alignment of S. flexneri IpaJ with members of C39-peptidase-like family identified by HHPred. IpaJ possesses invariant catalytic triad residues Cys 64, His 206 and Asp 218 (red). The conserved Gln 58, which helps form the oxyanion hole found in many proteases, is shown in green. Invariant residues and core hydrophobic residues found in the C39-family are shown in blue and magenta, respectively. Characterized C39-family member (GCN5-acyetyl transferase) and a C1-family member (Papain) are shown. b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm. c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source. d, Illustration of the yeast genomic clones isolated from the IpaJ suppressor screen. ARF1p, ARF2p and VPS15p are highlighted. e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.
Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ (2013)
f3

a–c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7). d, Structure of N-myristoylated ARF1 N terminus in untreated (left) and IpaJ-treated (right) samples. R group following Asn 3 denotes ARF1-strep protein residues 4–240. e, Reconstitution of ARF1-His N-myristoylation (N-myr) by NMTp in bacterial cells and IpaJ cleavage reaction in vitro (see Supplementary Information and Methods). f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain. g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel). h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.
Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ (2013)
f4

a, Multiple-sequence alignment of N-myristoylated glycine and downstream sequences from the indicated proteins (Hs, Homo sapiens; Sc, S. cerevisiae). b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide–eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS–polyacrylamide gel electrophoresis caused by proteolytic reaction. c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis. d, Fluorescence microscopy showing ARF1–mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm. e, Fluorescence microscopy showing ARF1–eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 1

Formation of Lewy body-like α-synuclein inclusions in cultured cells.

(A) Purified His-α-synuclein protein was agitated for 0–2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons. (B) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units. (C) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm. (D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 2

Autophagic clearance of α-synuclein inclusions.

(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes. (B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 3

The reduced incorporation of α-synuclein inclusions into autophagosomes by autophagy inhibition.

(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm. (B) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48–141 inclusions. Statistical analysis was performed with t-test (*p<0.01). (C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.01.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 4

Autophagic clearance of α-synuclein inclusions.

(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.05. This experiment was repeated three times. (B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction. (C) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm2) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.05, **p<0.001. (D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm. (E) Colocalization of α-synuclein inclusions (n = 41–73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.01.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 5

Involvement of p62 in the selective clearance of α-synuclein inclusions.

(A) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm. (B) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32–44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with t-test (*p<0.05). (C) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.01. This experiment was repeated three times. (D) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *p<0.01.
p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. (2012)
Figure 6

Impairment of mitochondria and mitophagy in cells harboring α-synuclein inclusions.

(A) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm. (B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 1

Figure 1. Ral and Rgl are required for salivary gland degradation

A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.

C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.

E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.

G Control animals (+/w; UAS-rglIR/+), n= 19, and those with salivary gland-specific knockdown of rgl (fkh-GAL4/w; UAS-rglIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 2

Figure 2. Loss of ral does not affect caspase activity during salivary gland degradation

A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.

B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments.

C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.

D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.

E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 3

Figure 3. ral is required for autophagy in dying salivary glands, but not for starvation-induced autophagy in fat body

A ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.

B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.

C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.

D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.

E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.

F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.

G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2)p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.

H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.

I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.

J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 4

Figure 4. The exocyst is not required for starvation-induced autophagy in fat body

A, C, E, G, I Fat bodies dissected from 4 hour starved animals expressing mCherry-Atg8a in all cells, and (A) sec5IR, (C) sec15IR, (E) sec3IR, (G) sec8IR, (I) exo84IR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.

B, D, F, H, J Quantification of data from (A, C, E, G, I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 5

Figure 5. The exocyst is required for autophagy in dying salivary gland cells

A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.

B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student’s t test (** p< 0.001, *** p< 0.0001).