EMBO/soda-nel huggingface dataset:
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 2

Figure 2. Vimentin is less dimethylated and involved in cancer invasion induced by MTAP deletion

A. The necessity of MTAP activity in cell invasion. CL1-5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05 compared to the vector control group). The protein expression levels of exogenous wild-type and mutant V5-MTAP were detected by Western blots.

B. Western blot analysis of the symmetric dimethylarginine (sDMA), monomethylarginine (mMA) and asymmetric dimethylarginine (aDMA) levels in MTAP-overexpressing and MTAP-knockout cells. V5-MTAP wild-type and D220A mutant were restored in H1650 MTAP-knockout cells. Data shown are representative of three independent experiments.

C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.

D. Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells. Arrow marks dimethyl-vimentin. Data shown are representative of three independent experiments.

E-F. Immunoprecipitation analysis for vimentin dimethylation in control and MTAP-knockout cells. Arrows mark vimentin, asterisks mark heavy chains, and arrowheads mark nonspecific bands. Data shown are representative of three independent experiments.

G. Vimentin-knockdown counteracts MTAP-loss-induced effect on cell invasion. MTAP-knockout and control H1650 cells were transfected with vimentin-specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). The silence efficiency of vimentin was examined by Western blots.
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 3

Figure 3. Suppression of vimentin sDMA level promotes cancer cell motility

A-D. Identification of symmetrically dimethylated arginine residues on vimentin by mass spectrometry analysis. MS/MS spectra evidence of sDMA at R196 (A), R207 (B), R345 (C), and R364 (D) of vimentin protein in CL1-5 cells. The sDMA-modified fragments with neutral loss 30.034 at the residue arginine (R) were annotated by blue color. Di, dimethyl at R; de, deamidated at N or Q; ox, oxidation at M; ++, fragment with double charge.

E. Illustration of vimentin protein domains and four dimethylated arginine residues are highlighted.

F. Validation of the PRMT5-catalyzed methylation sites on V5-vimentin. HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis. The intensity of sDMA signals was quantified by ImageJ software, normalized to the intensity of corresponding immunoprecipitated V5-vimentin, then relative to the V5-vimentin wild-type group and represented as percentage. Data were the averaged percentage from three independent experiments.

G. Single-cell tracking migration assays. H1650 control (sgCTRL) and vimentin-knockout (sgVIM) cells were transfected with GFP vector or GFP-vimentin wild-type, 4RK or 4RF mutant for 48 hours. Cells expressing GFP fluorescence were tracked by the time-lapse video microscopy system. Representative trajectories (left) and quantification of averaged velocity of cells (right, mean ± SD, Student t test, n=20, technical replicates, *p < 0.05). 4RK indicates R196/207/345/364K; 4RF indicates R196/207/345/364F. Data shown are representative of three independent experiments.
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 4

Figure 4. Vimentin protein level is upregulated by inhibition of PRMT5-mediated sDMA

A. Immunoﬂuorescence staining of V5-vimentin wild-type, 4RK and 4RF mutants expressed in CL1-0 cells. F-actin was stained with phalloidin, and nucleus was stained with DAPI. Scale bars, 10 μm. Data shown are representative of three independent experiments.

B. Reconstitution of vimentin-knockout cells with wild-type, 4RK- and 4RF-mutated V5-vimentin. Both protein and mRNA levels of vimentin were determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.

C. Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT-qPCR. Data shown are representative of three independent experiments.

D. Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.

E. Western blot analysis of vimentin protein levels in MTAP-knockout and control cells transfected with PRMT5 siRNAs (left) or Flag-PRMT5 (right). Arrows mark the site of Flag-PRMT5 and endogenous PRMT5. Data shown are representative of three independent experiments.

F. Effect of PRMT5 on the levels of ectopically expressed wild-type, 4RK- and 4RF-mutated V5-vimentin in HEK293T cells assessed by Western blots. Data shown are representative of three independent experiments.

G. Effects of wild-type and catalytically dead R368A mutant PRMT5 on the level of V5-vimentin in HEK293T cells detected by Western blot assays. Data shown are representative of three independent experiments.
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 5

Figure 5. Reduced sDMA levels stabilize vimentin

A. Measurement of V5-vimentin wild-type, 4RK and 4RF mutant protein levels in CL1-5 vimentin-knockout cells treated with 10 μM MG132 by Western blots. Data shown are representative of three independent experiments.

B. Western blot analysis of vimentin protein levels in control and MTAP-knockout cells treated with 10 μM MG132. Data shown are representative of three independent experiments.

C-D. Immunoprecipitation analysis of ubiquitination (C) and K48-linked polyubiquitination (D) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.

E. Immunoprecipitation analysis for K48-linked polyubiquitination of vimentin in CL1-0 control and MTAP-knockout cells. Arrow marks the site of vimentin. Data shown are representative of three independent experiments.

F-G. Western blot analysis of V5-vimentin wild-type, 4RK and 4RF mutant levels in CL1-0 cells (F) or endogenous vimentin levels in control and MTAP-knockout cells (G) treated with 50 μg/ml cycloheximide (CHX). Bottom: the intensity of V5-vimentin and vimentin signals was quantified by ImageJ software, normalized to the internal control β-actin, then normalized to zero time point, and plotted against time points (mean ± SE, n=3, biological replicates).
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 6

Figure 6. Negative association between MTAP/PRMT5 and vimentin is confirmed in lung cancer patients

A-B. The correlation of MTAP and vimentin or PRMT5 and vimentin at protein (A) and mRNA (B) levels from 89 lung adenocarcinoma patients. r is the Spearman's rank correlation coefficient.

C. Representative images of IHC staining.

D. An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman's rank correlation.

E. Kaplan-Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining. p value was obtained by log-rank test.
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 1

Figure 1. TRIM16 recruits IL-1β to LC3-positive carrier membranes for secretion.

(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1 were determined in supernatants. (B) THP-1 cells were subjected to GABARAP knockdown and processed as in A. (C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. (D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm. (E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. (F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU). (G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 2

Figure 2. Galectin-8 participates in IL-1β secretion and interacts with TRIM16.

(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS. (C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. (D) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells. (E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells. (F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm. (G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 3

Figure 3. TRIM16 recruits IL-1β to LC3-positive carrier membranes for secretion and interacts with Sec22b.

(A and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1 and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed – pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G. (C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1 secretion and treated with 1mM LLOMe for 1 h. (D) Quantification of analyses in (B). RI, relative intensity. (E) Whole cell lysates (immunoblots) analyzed in F and G. (F,G) Sucrose density gradient analysis of 25k membranes. Note that mature IL-1β (mIL-1β) is recruited to and co-fractionates with TRIM16+, Sec22b+, and LC3-II+ membranes in TRIM16wt HeLa cells reconstituted with pro-IL-1β and pro-caspase 1, but not in TRIM16KO mutant cells. (H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy. (J) Co-IP analysis between flag-TRIM16 with GFP-Sec22b or GFP-syntaxin-17 co-expressed in HEK293T cells. (K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe. (L) Co-IP analysis of interactions between deletion variants of flag-TRIM16 with GFP-Sec22b in HEK293T cells. (M and N) IL-1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b-interacting and its Sec22b-nonbinding mutant shown (O) Two color super-resolution image showing co-clustering of flag-TRIM16 (red) and GFP-Sec22b (green); Scale bar, 200 nm. (P) Cross-correlation analysis of flag-TRIM16 and GFP-Sec22b super-resolution data showing a characteristic separation of ~ 70 nm. Data, means  SEM; n≥5, except for immunoblot quantifications and image analyses where n≥3. *P < 0.05, (ANOVA or t test).
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 4

Figure 4. R-SNARE Sec22b participates in IL-1β secretion.

(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS. (C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. (D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h. (E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm. (F) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (ANOVA or t-test).
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 5

Figure 5. Qbc-SNAREs and plasma membrane Qa-SNAREs syntaxns-3 and -4 participate in IL-1β secretion.

(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B). (D) Secretory autophagy pathway. Upon lysosomal damage caused by inflammasome-agonists such as silica, alum, monosodium urate, and Leu-Leu-O-Me, inflammasome activation leads to caspase-1-dependent processing of cytosolic pro-IL1β into mature IL-1β. The autophagy regulatory systems (starvation/nutrition response systems: mTOR, LYNUS, TFEB) located at the lysosome transduce starvation signals (Napolitano & Ballabio, 2016b). Upon lysosomal damage and stress, galectin-8 recognizes -galactosides on the exposed lumenal membrane and/or glycosylated lysosomal lumenal proteins released into the cytosol and activates the galectin-8-TRIM16 complex. TRIM16 binds secretory autophagy cargo (mIL-1β and potentially other cargo). TRIM16 forms complexes with Sec22b to transfer the cargo to the autophagy-induced LC3-II+ membrane 25k carriers (a direct interaction between Sec22b and TRIM16 remains to be determined). Depiction of mIL-1β between the two membranes or within the lumen of the double membrane-delimited autophagosomal intermediates reflects the sequestration processes that may be involved: IL-1β is sequestered either through the reported Hsp90-dependent import across the delimiting membrane into the lumen of carriers topologically corresponding to the intermembarne space in conventional autophagosomes (Zhang et al., 2015), or is sequestered into the inner lumen of conventional double membrane autophagosomal organelles, specializing in secretion, simultaneously observed in the same study (Zhang et al., 2015). Not depicted is the compatibility of TRIM16’s receptor function with both of the above processes: i.e. either a hand-over of the cargo to Hsp90 or cargo sequestration via TRIM16 acting as a receptor/adaptor directly interacting with mAtg8s as previously reported (Mandell et al., 2014); the latter option is in keeping with the observations that TRIM16 is co-secreted with IL-1β (Munding et al., 2006). At this point, Sec22b molecules (acting as R-SNAREs) on the delimiting membrane facing the cytosol, carry out fusion at the plasma membrane in conjunction with the Qbc-SNAREs SNAP23 and SNAP29 and plasma membrane Qa-SNAREs syntaxins-3 and -4, thus delivering IL-1β to the extracellular milieu where it exerts its biological function. Data, means  SEM; n≥5. *P < 0.05, **P < 0.01, †P ≥ 0.05 (ANOVA or t-test).
Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy (2016)
Figure 6

Figure 6. Secretory autophagy plays a role in unconventional secretion of ferritin.

(A and B) Summary of candidate secretory autophagy cargos, identified by quantitative proteomics based on isobaric tandem mass tags (listed in Table EV1) and sorted by cellular compartments in (A) or presence/absence of leader signal peptide in (B). (C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe. (D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated. (E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe. (F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm; Data, means  SEM; n≥5. *P < 0.05, †P ≥ 0.05 (vs. LLOMe-treated or Mtb-infected scr control), (*)P < 0.05, (†)P ≥ 0.05 (vs. non-infected scr control; ANOVA or t-test).
An increase in Olfactory Bulb Neurogenesis Improves Discrimination of Highly
Similar Odorants (2018)
Figure 3

Fig 3. Integration and electrophysiological properties of 4D-derived granule cells.

(A) Experimental design to assess the integration of 4D-derived neurons.

(B) Fluorescence pictures (left) of immunolabeled GFP+RFP- (4D-) and GFP+RFP+ (4D+) apical dendrites of superficial granule neurons and quantifications (middle and right) of spine density and total dendritic length (box and whiskers) and 3D-Sholl (line graph profile) of apical dendrites starting from the soma (drawings) as the mean number of intersections at 10 μm intervals.

(C) 3D reconstruction of multi-channel confocal stacks acquired from a RFP+ apical dendrite of a granule cell showing co-localization with the pre- and post-synaptic markers VIAAT (granule neuron) and gephyrin (mitral cell), respectively (magnification shown in insets).

(D) Anti-RFP immunogold labeling of a cell in the superficial granule cell layer of a 4D+ mouse. Inset shows a representative RFP+ synapse (out of >10 analyzed from 4D+ mice).

(E-G) Current clamp recordings showing examples of spontaneous barrages of synaptic potentials (E), and repetitive spiking in response to depolarizing current steps (F-G) of 4D- (top) and 4D+ (bottom) mice. Inset in E (4D+ cell) is magnified (bottom). Note in G that the lag preventing spike initiation is longer at lower currents (green) and shorter at higher (black), suggesting the presence of an A-type K current typical of granule cells in 4D- vs. 4D+.

(H) Box and whiskers plots representing electrophysiological properties of neurons derived from 4D- and 4D+ NSC (black and red, respectively) including from left to right: spike number, resting membrane potential (Vrest), input resistance (Rin) and lag to spiking of the recorded superficial granule cells (see Fig. EV3 for additional parameters).

Data information: Data are presented as mean±SEM in the line graph in panel (B). No significant difference was found by unpaired Student's t-test (throughout) or repeated measures 2-way ANOVA (line graph in B). Boxplots in (B and H) shows the median (horizontal line), mean (+) and whiskers indicate the lowest and highest values within 1.5 interquartile range. Outliers were identified by Tukey's test. (B) N=3 mice, n>8 neurons per genotype; (E-H) N>5 mice, n=12 4D- and 10 4D+ neurons. Scale bars=5 µm (B and C), 1 µm (D and insets in C) and 0,5 µm (inset in D).
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 1

Mitochondrial network changes during muscle atrophy. (A) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Animals were starved for 24 h or denervated for 7 days. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm. (B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 2

FoxO3 controls mitochondrial network. (A) Imaging of FoxO3‐mediated modification of the mitochondrial network in soleus muscle of living mice. Adult soleus muscles were transfected with pDsRed2‐Mito and either c.a.FoxO3 or mock vector (control). Two weeks later, muscles were exposed and observed in situ using two‐photon microscopy. At least five animals per condition were studied. (B) Overexpression of c.a.FoxO3 induces mitochondrial changes and removal through autophagy. Muscle fibres were transfected by electroporation with YFP‐LC3, pDsRed2‐Mito and either c.a.FoxO3 or mock vector. Two weeks later, fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy. A higher magnification of the square is depicted on the right panels. White arrows point autophagosomes engulfing the mitochondria. (C) Overexpression of c.a.FoxO3 induces reduction in myofibre size and decrease in SDH staining. Left image: immunostaining for FoxO3 shows atrophic fibres, which over‐expressed FoxO3 (red) and are labelled by asterisks. Nuclei are stained by Hoechst (blue). Right image: histochemistry for succinate dehydrogenase (SDH) on serial section. Note that fibres positive for FoxO3 are negative for SDH staining (asterisks). (D) Electron micrograph of a control and c.a.FoxO3‐expressing fibres. Muscles were processed with standard fixation‐embedding procedure. Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria, respectively. Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres, whereas intermyofibrillar mitochondria are smaller and paler than control ones. Red arrowheads show a vesicle containing mitochondria. The image is representative of the FoxO3‐transfected fibres.