Journal: Journal of Cell Science

Article Title: Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification

doi: 10.1242/jcs.259324

Figure Lengend Snippet: nSMase2 regulates endolysosomal acidification by modulating V-ATPase assembly. (A) Confocal microscopy images of HeLa cells treated with DMSO or GW4869 (GW), co-stained for ceramide and ATP6AP2. Nuclei were stained with Hoechst. (B) Dot plots of mean fluorescence intensity of ceramide puncta (left) and mean fluorescence intensity of ceramide that colocalized with ATP6AP2 (right) from A. Data are presented as mean±s.d.; n >50 cells from two biological replicates. **** P <0.0001 (unpaired two-tailed Student's t -test). (C) Confocal microscopy images of the cytoplasmic distribution of ATP6V1E1 (V1E1) in HeLa cells upon SMPD3 KD (siSMPD3) compared with that of control cells (siCtrl). Actin was stained using phalloidin, and nuclei were stained using Hoechst. Images are representative of three experiments. (D) Confocal microscopy images of DMSO- and GW4869-treated HeLa cells co-stained for ATP6V0A1 and ATP6V1A. Nuclei were stained with Hoechst. Arrows indicate ATP6V0A1 and ATPV1A colocalization. The scale bar in the magnified inset represents 5 μm. (E) Dot plots showing colocalization analysis of ATP6V1A with ATP6V0A1 from D. Data are presented as mean±s.d.; n >20 from two biological replicates. **** P <0.0001 (unpaired two-tailed Student's t -test). (F) Western blot analysis of ATP6V0A1 (V0A1), ATP6V1A (V1A) and ATP6V1E1 (V1E1), as well as GAPDH as loading control, in total cell lysates (CX) and sEV fractions prepared from control (siCtrl) and SMPD3 KD (siSMPD3) HeLa cells. (G) Quantification of signal intensity of V0A1 in sEV fractions from F normalized to the corresponding loading control GAPDH in the CX before normalization to the respective control. Data are presented as mean±s.d. of three biological replicates. ** P <0.001 (unpaired two-tailed Student's t -test). (H) Western blot analysis of V0A1, V1A and V1E1 levels in organelle or cytosolic fractions upon SMPD3 KD in HeLa cells. Vinculin and GOSR2 were used as cytosolic or organelle markers, respectively. (I) Quantifications of signal intensity of V1A and V1E1 in organelle and cytosolic fractions, and V0A1 in the organelle fraction from H. Cytosolic and organelle signals for each subunit were normalized to their respective loading control, vinculin or GOSR2, before normalization to the control condition. Data are presented as mean±s.d. of three biological replicates. * P <0.01 (left, unpaired two-tailed Student's t -test; middle and right, one-way ANOVA followed by Sidak's multiple comparisons test). Molecular masses in D and H are indicated in kDa.

Article Snippet: The following antibodies and dilutions were used for western blotting (WB) or for immunofluorescence staining (IF): Alix (WB 1:1000; Santa Cruz, sc53540), CD63 (WB 1:100; IF 1:10; H5C6, AB_528158, Developmental Studies Hybridoma Bank), syntenin (WB 1:1000; IF 1:100; Abcam, ab133267), GAPDH (WB 1:1000; Millipore, CB1001), Calnexin (WB 1:1000; IF 1:100; Abcam, ab75801), HRS (IF 1:100; ProteinTech, 1039-1-AP), ceramide (IF 1:100; Enzo, ALX-804-196), LBPA (IF 1:100; Millipore, MABT837), nSMase2 (IF 1:100; Santa Cruz, sc-166637), ATP6V0A1 (WB 1:1000; IF 1:100; Novus Bio, NB1-89342), vinculin (WB 1:1000; ProteinTech, 66305-1-Ig), ATP6V1E1 (WB 1:1000; IF 1:100; ProteinTech, 15280-1-AP), ATP6V1A (WB 1:1000; Abnova, H00000523-M02), GOSR2 (WB 1:1000 ProteinTech, 66134-1-Ig), LAMP1 (WB 1:1000; IF 1:100; Abcam, ab24170).

Techniques: Confocal Microscopy, Staining, Fluorescence, Two Tailed Test, Control, Western Blot

Journal: Journal of Cell Science

Article Title: Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification

doi: 10.1242/jcs.259324

Figure Lengend Snippet: MVB cholesterol levels regulate sEV secretion by modulating V-ATPase assembly. (A) Confocal microscopy images of control (Ctrl) and U18666A (U18)-treated HeLa cells with intracellular acidic compartments labeled by Lysotracker. Nuclei were stained with Hoechst. (B) Quantifications of mean intensity of Lysotracker puncta (left) and Lysotracker staining puncta per cell from A. Data are presented as mean±s.d.; n >50 cells from three biological replicates. * P <0.01; **** P <0.0001 (unpaired two-tailed Student's t -test). (C) Western blot analysis of ATP6V0A1 (V0A1), ATP6V1A (V1A) and ATP6V1E1 (V1E1) levels in organelle or cytosolic fractions upon U18 treatment in HeLa cells. Vinculin and GOSR2 were used as the cytosolic and organelle markers, respectively. (D) Quantifications of signal intensity of V1A and V1E1 in organelle and cytosolic fractions, and V0A1 in the organelle fraction from C. Cytosolic and organelle signals for each subunit were normalized to their respective loading control, vinculin or GOSR2, before normalization to the control condition. (E) Western blot analysis of Alix, CD63, syntenin and CD81 in sEV fractions prepared from equal amounts of control or U18-treated HeLa cells. (F) Signal intensity quantifications of Alix, CD63, syntenin and CD81 in sEV fractions from E, calculated by normalizing the signal to loading control GAPDH levels in the corresponding cell lysates in G before normalization to their respective control. (G) Western blot analysis of the indicated exosomal markers in corresponding cell lysates (CX) from E. GAPDH was probed as a loading control. (H) Quantifications of intracellular Alix, CD63, syntenin and CD81 signal intensity from G normalized to loading control GAPDH before normalization to the respective control. (I) Representative size distribution of sEVs isolated from control and U18-treated HeLa cells. (J) NTA quantification of sEV concentration from I. The data shown in D,F,H and J represent the mean±s.d. of three biological replicates. * P <0.01; ** P <0.001 (unpaired two-tailed Student's t -test except for middle and right graphs in D where a one-way ANOVA with Sidak's multiple comparisons test was used). Molecular masses in C,E and G are indicated in kDa.

Article Snippet: The following antibodies and dilutions were used for western blotting (WB) or for immunofluorescence staining (IF): Alix (WB 1:1000; Santa Cruz, sc53540), CD63 (WB 1:100; IF 1:10; H5C6, AB_528158, Developmental Studies Hybridoma Bank), syntenin (WB 1:1000; IF 1:100; Abcam, ab133267), GAPDH (WB 1:1000; Millipore, CB1001), Calnexin (WB 1:1000; IF 1:100; Abcam, ab75801), HRS (IF 1:100; ProteinTech, 1039-1-AP), ceramide (IF 1:100; Enzo, ALX-804-196), LBPA (IF 1:100; Millipore, MABT837), nSMase2 (IF 1:100; Santa Cruz, sc-166637), ATP6V0A1 (WB 1:1000; IF 1:100; Novus Bio, NB1-89342), vinculin (WB 1:1000; ProteinTech, 66305-1-Ig), ATP6V1E1 (WB 1:1000; IF 1:100; ProteinTech, 15280-1-AP), ATP6V1A (WB 1:1000; Abnova, H00000523-M02), GOSR2 (WB 1:1000 ProteinTech, 66134-1-Ig), LAMP1 (WB 1:1000; IF 1:100; Abcam, ab24170).

Techniques: Confocal Microscopy, Control, Labeling, Staining, Two Tailed Test, Western Blot, Isolation, Concentration Assay

Journal: bioRxiv

Article Title: Monoacylglycerol disrupts Golgi structure and perilipin 2 association with lipid droplets

doi: 10.1101/2021.07.09.451829

Figure Lengend Snippet: Shown are OP9 cells treated with combinations of MAG, BFA and albumin. Cells were treated for 30 min with 40 μg/ml 2-Oleoylglycerol (MAG), 10 μg/ml BFA, and 2.2 % albumin, as indicated. Cells were stained with the Golgi markers anti-GM130 (shown as green) and anti-GS28 (shown as red) using 750 ng/ml and 2.5 μg/ml, respectively. Bar = 10 μm.

Article Snippet: Monoclonal antibody against mouse Golgi matrix protein GM130 was purchased from BD Biosciences; rabbit polyclonal antibody against Golgi SNARE protein (GS28 Antibody) from Proteintech™ (Chicago, IL, catalog number 16106-1-AP); rabbit polyclonal against β-COP from Thermo Scientific (catalog number PA1-061) and rabbit polyclonal antibody against calnexin from Enzo Life Sciences, Inc. (Farmingdale, NY, catalog number ADI-SPA-860).

Techniques: Staining

Journal: Frontiers in Physiology

Article Title: Agonists for Bitter Taste Receptors T2R10 and T2R38 Attenuate LPS-Induced Permeability of the Pulmonary Endothelium in vitro

doi: 10.3389/fphys.2022.794370

Figure Lengend Snippet: Protein expression levels of bitter taste receptors in human pulmonary endothelial cells.

Article Snippet: Whole cell ELISA was then performed using antibodies specific to the extracellular domain of VE-cadherin (BV6, amino acid unspecified; ), T2R10 (D-12, amino acid unspecified, sc-169,473), T2R14 (Boster BioPA5-39,710, amino acid 229–278) and T2R38 (Novus, NBP2-33711), and 488-fluorescent-conjugated secondary antibodies measured at 1 s exposure time at 485/552 ex/em using a fluorescent plate reader (Victor, Perkin Elmer).

Techniques: Expressing, Positive Control

Journal: Frontiers in Physiology

Article Title: Agonists for Bitter Taste Receptors T2R10 and T2R38 Attenuate LPS-Induced Permeability of the Pulmonary Endothelium in vitro

doi: 10.3389/fphys.2022.794370

Figure Lengend Snippet: Denatonium (Dena) and phenylthiourea (Phenyl) protect against LPS-induced permeability and VE-cadherin internalisation in human pulmonary arterial endothelial cells by acting directly through T2R10 and T2R38, respectively. Panels A and B: HPAEC were exposed to noscapine, denatonium or phenylthiourea (0.1 mm) in the presence (closed bars) or absence (open bars) of LPS (1 μg/ml) for 24 h. Monolayer permeability was measured using the FITC-dextran essay (panel A ) and VE-cadherin surface expression was assessed using whole cell ELISA (panel B ). Panels C and D: T2R10 (panel C )- and T2R38 (panel D )-specific siRNA was used to knockdown expression in HPAEC and confirmed using Western blot (inset) and whole cell ELISA [panels C (i) and D (i)]. Denatonium or phenylthiourea (0.1 mm) was added after 24 h in the presence (closed bars) or absence (open bars) of LPS (1 μg/ml) and monolayer permeability [panels C (ii) and D (ii)] and VE-cadherin surface expression [panels C (iii) and D (iii)] were studied. Data are presented as mean ± S.D. n = 6. Panel * p < 0.05 versus vehicle for LPS; # p < 0.05 versus vehicle for bitter agonists; δ p < 0.05 versus non-specific siRNA.

Article Snippet: Whole cell ELISA was then performed using antibodies specific to the extracellular domain of VE-cadherin (BV6, amino acid unspecified; ), T2R10 (D-12, amino acid unspecified, sc-169,473), T2R14 (Boster BioPA5-39,710, amino acid 229–278) and T2R38 (Novus, NBP2-33711), and 488-fluorescent-conjugated secondary antibodies measured at 1 s exposure time at 485/552 ex/em using a fluorescent plate reader (Victor, Perkin Elmer).

Techniques: Permeability, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Physiology

Article Title: Agonists for Bitter Taste Receptors T2R10 and T2R38 Attenuate LPS-Induced Permeability of the Pulmonary Endothelium in vitro

doi: 10.3389/fphys.2022.794370

Figure Lengend Snippet: Phenylthiourea (Phenyl), but not denatonium (dena), acts through a cAMP-Rac1-dependent signalling pathway to protect the pulmonary endothelial barrier. Panel A and C : HPAEC were exposed to noscapine, denatonium or phenylthiourea (0.1 mm) in the presence (closed bars) or absence (open bars) of LPS (1 μg/ml) for 24 h. cAMP (panel A ) and Rac1 (panel C ) levels were measured using commercially available ELISA kits. Panel B and D : T2R10 [panel B (i)]- or T2R38 [panel B (ii)]-specific siRNA was used to knockdown expression in HPAEC and denatonium or phenylthiourea (0.1 mm), respectively, was added after 24 h in the presence (closed bars) or absence (open bars) of LPS (1 μg/ml). cAMP (panel B ) and Rac1 (panel D ) levels were measured using commercially available ELISA kits. Panel E : HPAEC were exposed to the Rac1 inhibitor, NSC-23766, with phenylthiourea (0.1 mm) in the presence (closed bars) or absence (open bars) of LPS (1 μg/ml) for 24 h. Monolayer permeability was measured using the FITC-dextran essay [panel E (i)] and VE-cadherin surface expression was assessed using whole cell ELISA [panel E (ii)]. Data are presented as mean ± S.D. n = 5, 6. Panel * p < 0.05 versus vehicle for LPS; # p < 0.05 versus vehicle for NSC-23766.

Article Snippet: Whole cell ELISA was then performed using antibodies specific to the extracellular domain of VE-cadherin (BV6, amino acid unspecified; ), T2R10 (D-12, amino acid unspecified, sc-169,473), T2R14 (Boster BioPA5-39,710, amino acid 229–278) and T2R38 (Novus, NBP2-33711), and 488-fluorescent-conjugated secondary antibodies measured at 1 s exposure time at 485/552 ex/em using a fluorescent plate reader (Victor, Perkin Elmer).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Permeability

Journal: Nature Communications

Article Title: Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17

doi: 10.1038/s41467-025-55906-x

Figure Lengend Snippet: a , b Co-IP analysis of the endogenous HOPS subunits, STX17, VAMP7 and VAMP8 with HA-SNAP47 (WT, 3KR or 3KQ) in SNAP47 KO HeLa/Parkin cells ( a ). This result is part of Supplementary Fig . The proteins levels were quantified by ImageJ software and normalized with HA-SNAP47 ( b ). c , d HeLa cells stably expressing GFP-SNAP47 (WT, 3KR or 3KQ) were cultured in EBSS for 2 h, then stained with antibody against LC3 ( c ). Scale bar, 5 μm. The percentage of GFP-SNAP47-positive LC3 puncta in total LC3 puncta is shown ( d ). n = 3, 100 cells from three independent experiments. e , f HeLa/Parkin cells stably expressing mito-BFP and GFP-SNAP47 (WT, 3KR or 3KQ) were treated with OA for 4 h, then stained with with antibody against LC3 ( e ). Scale bar, 5 μm. The percentage of GFP-SNAP47 + LC3 + mito-BFP + puncta in total LC3 + mito-BFP + puncta is shown ( f ). n = 3, 100 cells from three independent experiments. g Schematic for the experimental procedures used in the reconstituted fusion reactions. h , i The effect of SNAP47 acetylation on fusion activity between proteoliposomes reconstituted with STX17 and VAMP8 ( h ). neg.ctrl. denotes no addition of SNAP29 and SNAP47. Statistics of the liposome fusion in ( h ) at 2400 s was shown ( i ). j , k The in vitro interaction of purified GST-SNAP47 (WT or mutants) with His-STX17 and VAMP8 ( j ) or His-STX17 and His-VAMP7 ( k ). l Schematic for the experimental procedures used in the SNARE complex assembly assay. m-o Assembly of the SNARE complex containing STX17, SNAP47 (WT, 3KR or 3KQ), and VAMP7/VAMP8 analyzed by FRET assays ( m , n ). Statistics of EPR (relative proximity ratio of FRET) at 2,500 s were shown ( o ). Data in ( b – o ) are presented as mean ± SD, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA ( b , o ) or one-way ANOVA ( d , f ). P values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Purified STX17 SNARE domain (residues 161−231, E198C), VAMP7 SNARE domain (residues 123−187, E152C), and VAMP8 SNARE domain (10−74, E39C) were conjugated with 3-fold molar ratio of ATTO647 maleimide (ATTO-TEC GmbH, #AD 550-45) and ATTO550 maleimide (ATTO-TEC GmbH, #AD 647N-45), respectively, according to the manufacturer’s instruction.

Techniques: Co-Immunoprecipitation Assay, Software, Stable Transfection, Expressing, Cell Culture, Staining, Activity Assay, In Vitro, Purification

Journal: Nature Communications

Article Title: Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17

doi: 10.1038/s41467-025-55906-x

Figure Lengend Snippet: a Co-IP analysis of interactions between the Flag-tagged HOPS subunits with HA-SNAP47 or HA-SNAP29 in HEK293T cells. PI line is for indicating the position of HA-SNAP47 and HA-SNAP29. n = 3 independent experiments. b Co-IP analysis of interactions between the endogenous HOPS subunits with Flag-SNAP47 or Flag-SNAP29 in HEK293T cells. n = 3 independent experiments. c In vitro interactions of purified GST-SNAP47 with His-tagged HOPS subunits. n = 3 independent experiments. d The central ionic layer of SNAP47 was identified through sequence alignment, participating residues glutamine-143 and glutamine-390 in SNAP47 are indicated by red color. The PH domain, SNARE motif, and second SNARE motif are colored by blue, green, and laurel-green, respectively. e, f Co-IP analysis of the endogenous HOPS subunits, STX17, VAMP7 and VAMP8 with HA-SNAP47 (WT or 2QR-Q143R/Q390R) in SNAP47 KO HeLa/Parkin cells ( e ). This result is part of Supplementary Fig . The proteins levels were quantified by ImageJ software and normalized with HA-SNAP47 ( f ). g – j WT and SNAP47 KO HeLa/Parkin cells were treated with OA for 4 h. Following separation via OptiPrep membrane flotation, autophagosome-enriched fractions (light-density fractions 3–5) were analyzed by immunoblotting using the indicated antibodies ( g ). The proteins levels were quantified by ImageJ software and normalized with LC3 ( h – j ). k – n WT, SNAP47 KO, and SNAP47 WT or Flag-SNAP47 LIR mutant complemented SNAP47 KO HeLa/Parkin cells were treated with OA for 4 h. Following separation via the OptiPrep membrane flotation, autophagosome-enriched fractions (light-density fractions 3–5) were analyzed by immunoblotting using the indicated antibodies ( k ). The proteins levels were quantified by ImageJ software and normalized with LC3 ( l – n ). Data in ( f – n ) are presented as mean ± SD, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA. P values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Purified STX17 SNARE domain (residues 161−231, E198C), VAMP7 SNARE domain (residues 123−187, E152C), and VAMP8 SNARE domain (10−74, E39C) were conjugated with 3-fold molar ratio of ATTO647 maleimide (ATTO-TEC GmbH, #AD 550-45) and ATTO550 maleimide (ATTO-TEC GmbH, #AD 647N-45), respectively, according to the manufacturer’s instruction.

Techniques: Co-Immunoprecipitation Assay, In Vitro, Purification, Sequencing, Software, Membrane, Western Blot, Mutagenesis