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  • 99
    Millipore bfa1
    A pH of ≤5.4 is essential for activating the GP64 fusogenicity needed for AcMNPV entry. (A) Fusogenicity of GP64 at different pHs. Sf9 cells were infected with Ac-BP egfp (MOI of 5 TCID 50 units/cell) and treated with PBS (pH as indicated) to trigger syncytium formation. Cell nuclei were counterstained (DAPI [4′,6-diamidino-2-phenylindole]; blue), and the syncytia are outlined (white dotted lines). The FITC channel (green) shows EGFP expression of infected cells. (B and C) HEK 293 cells (B) and Sf9 cells (C) transduced/infected with AcMNPV via the direct-fusion pathway as triggered by different pH values. Cells were incubated with Ac-BP egfp (MOI of 10 TCID 50 units/cell for HEK 293 cells or MOI of 0.2 TCID 50 units/cell for Sf9 cells) and treated with PBS (pH as indicated) to trigger the direct-fusion pathway. <t>BFA1</t> (20 nM) was used to block the endocytosis pathway. The percentages of transduced/infected HEK 293 and Sf9 cells were quantified by FCM at 24 h p.t. (HEK 293 cells) or 16 h p.i. (Sf9 cells). The results were normalized to those of the endocytosis pathway. All results are mean values ± SD ( n = 3 experimental replicates).
    Bfa1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    LC Laboratories bafilomycin a1
    ZKSCAN3 is required for autophagy inhibition induced by A30P α-synuclein. a ZKSCAN3 shRNA apparently inhibited ZKSCAN3 levels. b Representative immunoblots of lysates from midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a scramble (SCR) or ZKSCAN3 shRNA virus. Total protein extracts were immunoblotted for LC3, ZKSCAN3, TFEB or p62. c – g Shown is the densitometric quantification of corresponding protein levels described in b . h Midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a SCR or ZKSCAN3 shRNA virus. Then, neurons were treated with and without <t>Bafilomycin</t> A1 (BafA1, 10 nM) for 12 h. LC3-I/II levels were analyzed by immunoblotting. i The histogram plot shows the densitometric quantification of LC3-II levels. j Luciferase reporter assay for MAP1LC3B or GABARAPL2 constructs in midbrain dopaminergic neurons expressing A30P α-synuclein stably transfected either scramble (SCR) or ZKSCAN3-shRNA virus. Values are shown as mean ± SEM ( n = 3). Immunoblots are representative of n = 3. * P
    Bafilomycin A1, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 525 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology bafilomycin a1
    Inhibition of V-ATPase blocks ABCA1 mediated cholesterol efflux. ABCA1 induced (black bars) and non-induced (open bars) BHK cells were pretreated with 5 nM to 15 nM <t>bafilomycin</t> A1 for 16 hr ( A ), or 10 nM or 10 μM bafilomycin A1 for 1 hr ( B ), and % [ 3 H]cholesterol efflux was determined after a 4 hr chase +/− 5 μg/ml apoA1 (N=3; mean ± SD; groups with different letters above the bars show P ≤0.005 by ANOVA posttest). RAW264.7 cells were transfected with scramble or ATP6V 0 C siRNA by Nucleofection and ATP6V 0 C mRNA levels were measured by qPCR analysis normalized to β-actin mRNA ( C , N=3; mean ± SD; P
    Bafilomycin A1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Tocris bafilomycin a1
    Hyperosmolarity does not influence autophagic flux in NP cells. ( a ) Acridine orange staining of NP cells cultured under iso- (330 mOsm/kg H 2 O, top row) or hyperosmotic (500 mOsm/kg H 2 O, bottom row) condition, treated with (right) or without (left) <t>bafilomycin</t> A1. Hyperosmotic stimulus alone showed no change in number of acidic organelles. Bafilomycin A1 significantly reduced the acridine orange staining irrespective of osmolarity. Scale bar, 35 μm. ( b ) Quantification of acridine orange staining confirmed that hyperosmolarity had no effect on the number of acidic organelles. ( c ) LC3 immunofluorescence staining of NP cells cultured under hyperosmolarity with or without bafilomycin A1 treatment. Hyperosmotic stimulus did not upregulate LC3-positive autophagosomes. Scale bar, 20 μm. ( d ) Western blot analysis of NP cells cultured under increasing osmolarity (330–600 mOsm/kg H 2 O) with or without bafilomycin A1 treatment. The accumulation of LC3-II with bafilomycin A1 treatment was similar under iso- and hyperosmotic conditions. SQSTM1 also showed a trend of accumulation with bafilomycin A1 treatment under all conditions. The levels of ATG12-ATG5 and BECN1 remained unaltered between the experimental groups. ( e – h ) Densitometric analyses of multiple Western blots shown in ( d ). Bars represent mean ± SEM (n = 5). Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. NS, non-significant. BafA1, bafilomycin A1. Western blot images were cropped and acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images.
    Bafilomycin A1, supplied by Tocris, used in various techniques. Bioz Stars score: 97/100, based on 510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical bafilomycin a1
    MCU knockdown has no effect on SV exocytosis. Neurons transfected with syp-pHluorin and either shRNA targeting MCU ( shMcu1 and shMcu2 ) or a scrambled control ( shScr ) were stimulated with an action potential train (10 Hz, 120 s, indicated by the bar ) in the presence of <t>bafilomycin</t> A1. A , average traces for syp-pHluorin fluorescence (ΔF/F 0 ± S.E.) under each condition normalized to maximal fluorescence obtained in alkaline buffer (total SV recycling pool). B , average peak Syp-pHluorin fluorescence normalized to the total SV recycling pool. C, mean SV exocytosis rate calculated using a linear fit to Syp-pHluorin fluorescence during the first 12 s of stimulation. B and C , data are mean ± S.E. (shScr, n = 6; shMcu1, n = 7; shMcu2, n = 5; one-way ANOVA with Holm-Šídák post hoc tests; all non-significant; p > 0.05).
    Bafilomycin A1, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem bafilomycin a1
    SLC9 PROTAC Series (A) The chemical structure of the SLC degrader d9A-2. See Figure S5 A for structures of the related molecules. (B) HAP1 and KBM7 cells were treated with different concentrations of d9A-2. Within 8 h, degradation of SLC9A1 was observed in both cell lines. (C) Both WT and CRBN knockout KBM7 cell lines were treated with indicated concentrations of d9A-2 for 12 h. SLC9A1 degradation is observed in WT but not CRBN knockout. (D) Chemical “rescue” of d9A-2-driven degradation. WT KBM7 cell lines were treated with 0.5 μM d9A-2 for 16 h. These cells were also treated with bortezomib (bort.) (0.25 μM), MG-132 (MG) (1 μM), MLN4924 (MLN) (1 μM), pomalidomide (poma.) (1 μM), or <t>bafilomycin</t> A1 (bafi.) (10 μM). All molecules, apart from bafilomycin, could rescue SLC9A1 from degradation. (E) Selectivity of d9A-2 was tested across HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SLC9B1, and SLC9B2. Cells were treated with varying concentrations of d9A-2 for 16 h, after which degradation of the exogenous SLCs was monitored. At 0.25 μM, SLC9A1 is the only SLC9 member that is completely degraded. (F) Kinetics of d9A-2-induced degradation tested in HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A4, and SLC9A6. d9A-2 (0.75 μM) was added to HAP1 cell lines for the indicated length of time. SLC9A1 is the only protein that is mostly degraded after 6 h.
    Bafilomycin A1, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AG Scientific bafilomycin a1
    ATP6V0C regulation of autophagy-lysosome pathway markers. Representative western blots for lysosome marker LAMP-1 (A) or autophagosome marker LC3-II (B) from lysates collected following nucleofection with Non-target or ATP6V0C siRNA and subsequent treatment for 48 h with 0–100 nM <t>bafilomycin</t> A1 (BafA1). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels C–F. Within group comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for LC3-II (C) or LAMP-1 (D) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p
    Bafilomycin A1, supplied by AG Scientific, used in various techniques. Bioz Stars score: 92/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen bafilomycin a1
    GAS effector Nga inhibits starvation-induced LC3-puncta formation. Confocal micrographs (a) and quantification of starvation-induced LC3-puncta formation (b) and GcAV formation (c and d). HeLa cells stably expressing GFP-LC3 infected with GAS wild-type or its mutants for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Cells were fixed and immunostained for GAC (group A carbohydrate) to observe GAS (red). (e and f) Conversion of LC3-I to LC3-II in response to starvation during GAS infection. HeLa cells were infected with indicated GAS strains for 2 h and incubated with regular (control) or starvation medium for 1 h with or without <t>bafilomycin</t> A1. Representative WB images of LC3 and GAPDH (control) (E) and quantification of LC3 conversion (LC3-I to LC3-II) (f). Intensity ratio of LC3-II:LC3-I were normalized to that in non-infected (NI) cells without bafilomycin A1. (g) Confocal micrographs of starvation-induced LC3-puncta formation. HeLa cells stably expressing GFP-LC3 infected with GAS Nga R289K,G330D for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Scale bars: 10 μm. Data in (b, c, d, and f) are mean ± SEM of 3 independent experiments. Data were tested by two-tailed Student’s t-test: *** P
    Bafilomycin A1, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals bafilomycin a1
    Autophagy modulates the sensitivity of GBM primary cells to chemotherapy. a The effects of autophagy to chemoresistance were confirmed with primary cells from GBM patients. Similar to what was observed with GBM cell lines, upon 5-day drug treatment, while autophagy inhibition by <t>bafilomycin</t> A1 sensitized the primary cancer cells to death, autophagy induction by rapamycin dramatically attenuated the cytotoxicity of chemotherapy and promoted cancer cell survival. b As determined by the AAV-mRFP-GFP-LC3B reporter assay, for all the groups, most of the surviving cells manifested high level of autophagic activity after 5-day treatment, suggesting that autophagy underlies the chemoresistance of GBM primary cells
    Bafilomycin A1, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bafilomycin a1 from streptomyces griseus
    Lipid loading does not affect macrophage autophagy: ex vivo and in vitro studies A. Neutral lipid species in total eWAT ATMs of LFD and HFD mice were detected by LipidTox ™ staining. A representative image is shown. B. Quantification of neutral lipid species represented in A, n=15 mice/group. C. Neutral lipid species in BMDMs treated for 24 h with 500 μM palmitate and 500 μM oleate were detected by LipidTox ™ staining. A representative image is shown. D. Quantification of neutral lipid species represented in C, BMDMs from n=4 mice/group. E – J. The ratio of LC3-II:LC3-I or levels of p62 normalized to tubulin is reported by Western blotting in BMDMs treated with the indicated fatty acid for 24 h from n=4 mice/group. Where indicated, BMDMs were treated with 10 μm <t>bafilomycin</t> for the last 3 h in the 24 h fatty acid treatment time period. F and G are quantifications of E. I and J and quantifications of H. FMO = fluorescence minus one, Palm = palmitate, Baf = bafilomycin * = p
    Bafilomycin A1 From Streptomyces Griseus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bafilomycin a1 baf
    Lysosomal cysteine proteases activate PEDV pseudovirus entry. A and B , Huh-7 cells ( A ) or PK-15 cells ( B ) were preincubated with <t>bafilomycin</t> A1 ( Baf-A1 , a lysosomal acidification inhibitor) or E-64d (a lysosomal cysteine protease inhibitor) at the indicated concentrations and then transduced by PEDV pseudoviruses. C and D , retroviruses pseudotyped with VSV envelop glycoprotein ( i.e. VSV pseudoviruses) were used as a control. The pseudovirus entry in target cells without any inhibitor treatment was taken as 100%. Error bars indicate S.E. ( n = 5).
    Bafilomycin A1 Baf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM bafilomycin a1
    High glucose (HG) stimulates autophagic activity with downstream suppression of autophagic flux in vitro . Autophagic flux in proximal tubular epithelial cells (PTECs) during normal and HG treatment was estimated by the conversion from microtubule-associated protein 1 light chain 3-I (MAP1LC3-I) to MAP1LC3-II as a readout of autophagosome formation ( n =4–5, respectively). (A) Autophagic flux index (defined as the proportion of the levels of MAP1LC3-II in the presence of <t>bafilomycin</t> A1 (BafA1) to the levels of MAP1LC3-II in the absence of BafA1) is calculated at the indicated times. (B) Effect of insulin on autophagic flux in PTECs during HG exposure was assessed. Representative immunoblots are shown. Data are presented as means±SEM. Statistically significant differences are indicated. ACTB, actin, beta; LG, low glucose. * P
    Bafilomycin A1, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam bafilomycin a1
    Chemical Inhibition of VCP ATPase Activity with ML240 Impairs IFITM3 Turnover, Lysosomal Sorting, and Trafficking (A) Analysis of IFITM3 and ubiquitination levels upon treatment with ML240. HEK293T cells were transfected with HA-IFITM3 for 12 h, treated with DMSO or ML240 (2.5 μM) for another 12 h, and then lysed for western blot analysis. The red asterisks indicate IFITM3 ubiquitination bands with high molecular weights. (B) Analysis of IFITM3 turnover upon treatment with ML240. HeLa cells expressing HA-IFITM3 were treated with CHX (25 μg/mL) for the indicated times in the presence of DMSO or ML240 (2.5 μM) and lysed for western blot analysis. (C) Quantification of IFITM3 levels normalized to tubulin levels shown in (B). Data are represented as mean ± SD, n = 3. (D) Immunofluorescence analysis of HA-IFITM3 in the presence of <t>bafilomycin</t> A1 or ML240. HeLa cells were transfected with HA-IFITM3 and mCherry-LAMP1, treated with DMSO, bafilomycin A1 (100 ng/mL), or ML240 (2.5 μM) for 12 h, and processed for immunofluorescence with an Alexa Fluor 488-conjugated anti-HA antibody. Scale bars, 10 μm. The right panels show magnified squared regions in the corresponding left panels. White arrows indicate enlarged IFITM3-containing compartments. (E) Relative percentage of DiD-IAV particles colocalized with IFITM3 at the time of dequenching upon ML240 treatment. HeLa IFITM2/3-KO cells expressing BODIPY-labeled IFITM3 were infected with DiD-IAV particles, acutely treated with ML240 (1 μM), and monitored for DiD dequenching and IFITM3 trafficking by time-lapse imaging. Data are represented as mean ± SD of three independent experiments. ∗∗∗∗p
    Bafilomycin A1, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bafilomycin a1
    Detection of autophagy-related markers. (A) Western blot analysis of p62 and LC3-II in DLD-1 scr and DLD-1 KOAGR2 cells in response to treatments as indicated. (B) Changes in the LC3-II level following co-treatment with <t>bafilomycin</t> A1. (C) Western blot
    Bafilomycin A1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA bafilomycin a1
    Increased NAA pathway activity elevated the number of lysosomes and induced autophagy. Mature control and Nat8l o/e iBACs were used for the analysis. (A) Immunoblot of nuclear fraction showing transcription factor EB (TFEB) translocation to the nuclei. Histone H3 was used as loading control for nucleus fraction ( n = 3). (B) Representative electron micrographs of control and Nat8l o/e iBACs and (C) corresponding number of lysosomes, counted from all 90 electron micrographs from 3 biological replicates. Scale bar = 1 μm. L: lysosome, LD: lipid droplet, M: mitochondria N: nucleus. (D) Representative confocal image of iBACs stained with LysoTracker Red ( n = 3). (E) Number of Lysotracker Red stained lysosomes counted by flow cytometry ( n = 3). (F) Autophagic flux was monitored by immunoblotting against LC3 in the presence or absence of autophagy inhibitor <t>bafilomycin</t> A1 (BafA1) GAPDH was used as loading control ( n = 3). (G) Representative confocal images of cells transfected with tandem mRFP-GFP-LC3 vector. GFP and mRFP channels are shown in green and red, respectively. Elevated number of autolysosomes (red puncta in merged image) indicates increased autophagic flux. (H) Immunoblot showing LC3 in cell lysates with or without acetate supplementation to growth media (48 h, 10 mM NaAc) ( n = 3). (I) Densitometry of LC3-II bands from (I) normalized to ACTb ( n = 3). Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t -test (* p
    Bafilomycin A1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bafa1
    Degradation profiles of Golgi-localized model QC substrates. (A) Domain outline of Golgi QC substrates targeted to the cis- (MAN2A1-EGFP-HT2) and trans- (ST6GAL1- and B4GT-EGFP-HT2) Golgi. (B) Flow cytometry analysis quantifying the levels of MAN2A1-EGFP-HT2 in HEK293 cells on the indicated treatments for 6 h (EPX, epoxomicin; <t>BafA1,</t> bafilomycin A). CHX was added 1 h prior to the indicated treatments. (C) Same analysis as in B for HEK293 cells expressing B4GT-EGFP-HT2 or ST6GAL1-EGFP-HT2 ( n = 2, data represent mean ± SEM). (D) Flow cytometry analysis quantifying the levels of B4GT-EGFP-HT2 in HeLa cells on the indicated treatments for 6 h in the absence (left) or presence (right) of CHX ( n = 3, data represent mean ± SEM, results of t test are shown).
    Bafa1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kamiya bafilomycin a1
    The 7B2-proPC2 complex is not accumulated in the ER but rapidly transported to the Golgi. AtT-20 cells expressing PC2 and 7B2 were labeled for 10 min and chased for the indicated times in the presence of 1 μM <t>bafilomycin</t> A1. PC2 and 7B2 were immunoprecipitated under native conditions, denatured, and digested with endoglycosidase H.
    Bafilomycin A1, supplied by Kamiya, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adipogen bafilomycin a1
    Acidic pH causes a modest increase in RNAIII expression in vitro . (A) Phagosomal pH was evaluated in THP-1 macrophages treated with or without the phagosomal acidification inhibitor <t>bafilomycin</t> A1 (Baf; 100 nM) or NH 4 Cl (40 mM) and infected with Oregon
    Bafilomycin A1, supplied by Adipogen, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH bafilomycin a1
    Validation of GABARAPL2 endoHA /ACSL3 endoNeonGreen cells. A, GABARAPL2 endoHA and GABARAPL2 endoHA /ACSL3 endoNeonGreen cells as well as parental HeLa cells transiently transfected with TOMM20-NeonGreen were lysed and analyzed by immunoblotting with indicated antibodies. B, Fixed GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were immunostained with an anti-calnexin antibody. Scale bar: 10 µm. C, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were treated with oleic acid or EtOH (control) for 24 hrs followed by fixation and immunolabeling of phospholipids and neutral lipids. Scale bar: 10 µm. Two confocal planes are shown for oleic acid treatment. D, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells treated with Torin1 and <t>BafA1</t> or ATG7 inhibitor were fixed and immunolabeled with an anti-HA antibody. Scale bar: 10 µm. Arrowheads indicate HA-NeonGreen colocalization events.
    Bafilomycin A1, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co bafilomycin a1
    Sym004 promotes ubiquitin-independent, ESCRT-dependent lysosomal degradation of EGFR. (a) SCC47 cells were serum-starved for 1 h, then MG132 (10 µM) or <t>bafilomycin</t> A1 (600 nM) were added, followed 1 h later by cisplatin (50 µM), cetuximab, Sym004 or EGF. The cells were incubated overnight and lysed with RIPA buffer. (b) SCC47 cells were serum-starved for 1 h, then bafilomycin A1 (600 nM) was added followed 1 h later by Sym004-AF568 or EGF-AF647. The cells were incubated overnight, fixed and stained with EGFR-AF488 antibody. (c) Serum-starved SCC47 were treated with cetuximab, Sym004 or EGF as indicated, then lysed. EGFR was immunoprecipitated with anti-EGFR antibody (normal mouse IgG used in Control). For quantifications, ubiquitylated EGFR was normalised to total EGFR. (d) Serum-starved, Hrs- or Tsg101-depleted (or control) SCC47 cells were treated with Sym004 (3 µg/ml or 30 µg/ml) or EGF for 6 h, then lysed. (e) The cells treated as in (d) were fixed and stained with EGFR-AF488. Nuclei were stained with Hoechst 33342. All immunoblots were cropped for clarity.
    Bafilomycin A1, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Applichem bafa1
    Modulation of nonclassical secretory pathway by <t>BafA1</t> treatment in H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T. ( A ) Scheme of markers for distinct nonclassical secretory pathways including exosome release, endocytic recycling pathway, and microparticle shedding. MVBs = multivesicular bodies, ILVs = intraluminal vesicles, PS = phosphatidylserine ( B ) Quantification of extracellular CD63 levels in the medium of H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP compared to control cells (mock) by dot blot analysis. Cells have been treated with or without 200 nM BafA1 for 12 h. ( C ) Quantification of RAB11A expression in H4 cell lysates expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP -/+ BafA1 treatment. ( D ) Left: FACS analysis of released microparticles by transfected H4 cells -/+ BafA1 using a FITC-labeled ANXA5 antibody. Right: Representative scatter plots and histograms showing medium with ANXA5 as control for background signals, unstained microparticles to exclude auto fluorescence and microparticles stained for ANXA5 as positive control defining gating criteria. All values are mean + s.e.m; differences are significant at (*) P
    Bafa1, supplied by Applichem, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A pH of ≤5.4 is essential for activating the GP64 fusogenicity needed for AcMNPV entry. (A) Fusogenicity of GP64 at different pHs. Sf9 cells were infected with Ac-BP egfp (MOI of 5 TCID 50 units/cell) and treated with PBS (pH as indicated) to trigger syncytium formation. Cell nuclei were counterstained (DAPI [4′,6-diamidino-2-phenylindole]; blue), and the syncytia are outlined (white dotted lines). The FITC channel (green) shows EGFP expression of infected cells. (B and C) HEK 293 cells (B) and Sf9 cells (C) transduced/infected with AcMNPV via the direct-fusion pathway as triggered by different pH values. Cells were incubated with Ac-BP egfp (MOI of 10 TCID 50 units/cell for HEK 293 cells or MOI of 0.2 TCID 50 units/cell for Sf9 cells) and treated with PBS (pH as indicated) to trigger the direct-fusion pathway. BFA1 (20 nM) was used to block the endocytosis pathway. The percentages of transduced/infected HEK 293 and Sf9 cells were quantified by FCM at 24 h p.t. (HEK 293 cells) or 16 h p.i. (Sf9 cells). The results were normalized to those of the endocytosis pathway. All results are mean values ± SD ( n = 3 experimental replicates).

    Journal: Journal of Virology

    Article Title: The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

    doi: 10.1128/JVI.00709-19

    Figure Lengend Snippet: A pH of ≤5.4 is essential for activating the GP64 fusogenicity needed for AcMNPV entry. (A) Fusogenicity of GP64 at different pHs. Sf9 cells were infected with Ac-BP egfp (MOI of 5 TCID 50 units/cell) and treated with PBS (pH as indicated) to trigger syncytium formation. Cell nuclei were counterstained (DAPI [4′,6-diamidino-2-phenylindole]; blue), and the syncytia are outlined (white dotted lines). The FITC channel (green) shows EGFP expression of infected cells. (B and C) HEK 293 cells (B) and Sf9 cells (C) transduced/infected with AcMNPV via the direct-fusion pathway as triggered by different pH values. Cells were incubated with Ac-BP egfp (MOI of 10 TCID 50 units/cell for HEK 293 cells or MOI of 0.2 TCID 50 units/cell for Sf9 cells) and treated with PBS (pH as indicated) to trigger the direct-fusion pathway. BFA1 (20 nM) was used to block the endocytosis pathway. The percentages of transduced/infected HEK 293 and Sf9 cells were quantified by FCM at 24 h p.t. (HEK 293 cells) or 16 h p.i. (Sf9 cells). The results were normalized to those of the endocytosis pathway. All results are mean values ± SD ( n = 3 experimental replicates).

    Article Snippet: For direct-fusion pathway-mediated transduction, HeLa cells were treated with 20 nM BFA1 (Sigma-Aldrich) at 37°C for 30 min, chilled on ice, and incubated with Ac-BP egfp (MOI of 10 TCID50 units/cell) at 4°C for 1 h. The virus-bound cells were collected to measure the total bound virions or treated with PBS (pH 4.8) for 5 min. After that, cells were cultured with DMEM containing BFA1 at 37°C for 1 h. Then, cells were digested by 0.05% (wt/vol) trypsin for 10 min, washed with PBS, and subjected to DNA extraction.

    Techniques: Infection, Expressing, Incubation, Blocking Assay

    Two endosomal acidification inhibitors reveal the relatively high pH of the endosome where AcMNPV fuses in mammalian cells. (A to C) Effects of BFA1 and NH 4 Cl on virus entry. Sf9 and HeLa cells were incubated with BFA1 or NH 4 Cl at the indicated concentrations. Sf9 (A) and HeLa (B) cells were infected/transduced by Ac-BP egfp (MOI of 0.2 TCID 50 units/cell for Sf9 cells or MOI of 10 TCID 50 units/cell for HeLa cells). (C) HeLa cells were transduced by VSV-GFP. Cells were analyzed by FCM for EGFP expression at 24 h p.t. (HeLa cells) or 16 p.i. (Sf9 cells). (D and E) Effects of BFA1 and NH 4 Cl on virus-endosome fusion. Sf9 (D) and HeLa (E) cells were incubated with 20 nM BFA1 or 20 mM NH 4 Cl and infected/transduced by Ac-DiD (MOI of 200 TCID 50 units/cell). Cells were imaged at 60 min p.i./p.t. The fluorescence intensities of DiD were analyzed, and the percentages of dequenched DiD were calculated. (F) BFA1 and NH 4 Cl increased endosomal pH. HeLa cells were incubated with 20 nM BFA1 or 20 mM NH 4 Cl and stained with LSYB. Cells were imaged via fluorescence microscopy, and the pH of each endosome was calculated by ratiometric fluorescence measurement. Five randomly selected fields were analyzed per group. For panels A to E, the results were normalized to those of cells not treated with BFA1 and NH 4 Cl. For panels A to C, all results are mean values ± SD ( n = 3 experimental replicates). For panels D and E, the results are mean values ± SD ( n = 2 independent experiments). Statistical analysis was performed using the unpaired t test: *, P

    Journal: Journal of Virology

    Article Title: The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

    doi: 10.1128/JVI.00709-19

    Figure Lengend Snippet: Two endosomal acidification inhibitors reveal the relatively high pH of the endosome where AcMNPV fuses in mammalian cells. (A to C) Effects of BFA1 and NH 4 Cl on virus entry. Sf9 and HeLa cells were incubated with BFA1 or NH 4 Cl at the indicated concentrations. Sf9 (A) and HeLa (B) cells were infected/transduced by Ac-BP egfp (MOI of 0.2 TCID 50 units/cell for Sf9 cells or MOI of 10 TCID 50 units/cell for HeLa cells). (C) HeLa cells were transduced by VSV-GFP. Cells were analyzed by FCM for EGFP expression at 24 h p.t. (HeLa cells) or 16 p.i. (Sf9 cells). (D and E) Effects of BFA1 and NH 4 Cl on virus-endosome fusion. Sf9 (D) and HeLa (E) cells were incubated with 20 nM BFA1 or 20 mM NH 4 Cl and infected/transduced by Ac-DiD (MOI of 200 TCID 50 units/cell). Cells were imaged at 60 min p.i./p.t. The fluorescence intensities of DiD were analyzed, and the percentages of dequenched DiD were calculated. (F) BFA1 and NH 4 Cl increased endosomal pH. HeLa cells were incubated with 20 nM BFA1 or 20 mM NH 4 Cl and stained with LSYB. Cells were imaged via fluorescence microscopy, and the pH of each endosome was calculated by ratiometric fluorescence measurement. Five randomly selected fields were analyzed per group. For panels A to E, the results were normalized to those of cells not treated with BFA1 and NH 4 Cl. For panels A to C, all results are mean values ± SD ( n = 3 experimental replicates). For panels D and E, the results are mean values ± SD ( n = 2 independent experiments). Statistical analysis was performed using the unpaired t test: *, P

    Article Snippet: For direct-fusion pathway-mediated transduction, HeLa cells were treated with 20 nM BFA1 (Sigma-Aldrich) at 37°C for 30 min, chilled on ice, and incubated with Ac-BP egfp (MOI of 10 TCID50 units/cell) at 4°C for 1 h. The virus-bound cells were collected to measure the total bound virions or treated with PBS (pH 4.8) for 5 min. After that, cells were cultured with DMEM containing BFA1 at 37°C for 1 h. Then, cells were digested by 0.05% (wt/vol) trypsin for 10 min, washed with PBS, and subjected to DNA extraction.

    Techniques: Incubation, Infection, Expressing, Fluorescence, Staining, Microscopy

    The fusogenicity of GP64 is strictly associated with the transduction efficiency in mammalian cells. (A) Fusogenicity of GP64 mutants. Sf9 cells were infected with recombinant AcMNPVs (MOI of 5 TCID 50 units/cell) for 48 h, and the syncytium formation assay was performed. Fusogenicity was determined by dividing the number of nuclei in syncytia by the total number of cells in a field. Data were normalized to those of WT virus. (B) One-step growth curves of the various recombinant viruses. (C) HEK 293 cells were transduced with recombinant viruses at an MOI of 10 TCID 50 units/cell and imaged for EGFP expression at 24 h p.t. (D) HEK 293 cells transduced with recombinant viruses as described in the legend to panel C were analyzed by FCM. Data were normalized to those of the WT virus. (E) Correlation of transduction efficiency and fusogenicity. Linear regression analysis was performed, and the trend line is displayed on the scatter plot, for which the function is Y = 0.8345 × X + 11.79 ( R 2 = 0.96). (F) The transduction of GP64 D301E mutation-containing virus was rescued by a low-pH trigger. Cells were incubated with D301E or WT virus at 4°C and treated with PBS (pH as indicated) to trigger the direct-fusion pathway. BFA1 (20 nM) was used to block the endocytosis pathway. The percentage of EGFP-positive cells transduced with D301E virus was divided by that of cells transduced with the WT virus at each pH. (G) Low-pH trigger increases the transduction of recombinant viruses. HEK 293 cells were transduced with recombinant viruses through the endocytosis or direct-fusion (triggered with pH 4.8) pathway as described above. The results were normalized to those of WT transduction through the endocytosis pathway. For panels A, B, D, F, and G, all results are mean values ± SD ( n = 3 experimental replicates). Statistical analysis was performed using the unpaired t test: *, P

    Journal: Journal of Virology

    Article Title: The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

    doi: 10.1128/JVI.00709-19

    Figure Lengend Snippet: The fusogenicity of GP64 is strictly associated with the transduction efficiency in mammalian cells. (A) Fusogenicity of GP64 mutants. Sf9 cells were infected with recombinant AcMNPVs (MOI of 5 TCID 50 units/cell) for 48 h, and the syncytium formation assay was performed. Fusogenicity was determined by dividing the number of nuclei in syncytia by the total number of cells in a field. Data were normalized to those of WT virus. (B) One-step growth curves of the various recombinant viruses. (C) HEK 293 cells were transduced with recombinant viruses at an MOI of 10 TCID 50 units/cell and imaged for EGFP expression at 24 h p.t. (D) HEK 293 cells transduced with recombinant viruses as described in the legend to panel C were analyzed by FCM. Data were normalized to those of the WT virus. (E) Correlation of transduction efficiency and fusogenicity. Linear regression analysis was performed, and the trend line is displayed on the scatter plot, for which the function is Y = 0.8345 × X + 11.79 ( R 2 = 0.96). (F) The transduction of GP64 D301E mutation-containing virus was rescued by a low-pH trigger. Cells were incubated with D301E or WT virus at 4°C and treated with PBS (pH as indicated) to trigger the direct-fusion pathway. BFA1 (20 nM) was used to block the endocytosis pathway. The percentage of EGFP-positive cells transduced with D301E virus was divided by that of cells transduced with the WT virus at each pH. (G) Low-pH trigger increases the transduction of recombinant viruses. HEK 293 cells were transduced with recombinant viruses through the endocytosis or direct-fusion (triggered with pH 4.8) pathway as described above. The results were normalized to those of WT transduction through the endocytosis pathway. For panels A, B, D, F, and G, all results are mean values ± SD ( n = 3 experimental replicates). Statistical analysis was performed using the unpaired t test: *, P

    Article Snippet: For direct-fusion pathway-mediated transduction, HeLa cells were treated with 20 nM BFA1 (Sigma-Aldrich) at 37°C for 30 min, chilled on ice, and incubated with Ac-BP egfp (MOI of 10 TCID50 units/cell) at 4°C for 1 h. The virus-bound cells were collected to measure the total bound virions or treated with PBS (pH 4.8) for 5 min. After that, cells were cultured with DMEM containing BFA1 at 37°C for 1 h. Then, cells were digested by 0.05% (wt/vol) trypsin for 10 min, washed with PBS, and subjected to DNA extraction.

    Techniques: Transduction, Infection, Recombinant, Tube Formation Assay, Expressing, Mutagenesis, Incubation, Blocking Assay

    ZKSCAN3 is required for autophagy inhibition induced by A30P α-synuclein. a ZKSCAN3 shRNA apparently inhibited ZKSCAN3 levels. b Representative immunoblots of lysates from midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a scramble (SCR) or ZKSCAN3 shRNA virus. Total protein extracts were immunoblotted for LC3, ZKSCAN3, TFEB or p62. c – g Shown is the densitometric quantification of corresponding protein levels described in b . h Midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a SCR or ZKSCAN3 shRNA virus. Then, neurons were treated with and without Bafilomycin A1 (BafA1, 10 nM) for 12 h. LC3-I/II levels were analyzed by immunoblotting. i The histogram plot shows the densitometric quantification of LC3-II levels. j Luciferase reporter assay for MAP1LC3B or GABARAPL2 constructs in midbrain dopaminergic neurons expressing A30P α-synuclein stably transfected either scramble (SCR) or ZKSCAN3-shRNA virus. Values are shown as mean ± SEM ( n = 3). Immunoblots are representative of n = 3. * P

    Journal: Cell Death & Disease

    Article Title: A30P mutant α-synuclein impairs autophagic flux by inactivating JNK signaling to enhance ZKSCAN3 activity in midbrain dopaminergic neurons

    doi: 10.1038/s41419-019-1364-0

    Figure Lengend Snippet: ZKSCAN3 is required for autophagy inhibition induced by A30P α-synuclein. a ZKSCAN3 shRNA apparently inhibited ZKSCAN3 levels. b Representative immunoblots of lysates from midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a scramble (SCR) or ZKSCAN3 shRNA virus. Total protein extracts were immunoblotted for LC3, ZKSCAN3, TFEB or p62. c – g Shown is the densitometric quantification of corresponding protein levels described in b . h Midbrain dopaminergic neurons expressing A30P α-synuclein were infected with either a SCR or ZKSCAN3 shRNA virus. Then, neurons were treated with and without Bafilomycin A1 (BafA1, 10 nM) for 12 h. LC3-I/II levels were analyzed by immunoblotting. i The histogram plot shows the densitometric quantification of LC3-II levels. j Luciferase reporter assay for MAP1LC3B or GABARAPL2 constructs in midbrain dopaminergic neurons expressing A30P α-synuclein stably transfected either scramble (SCR) or ZKSCAN3-shRNA virus. Values are shown as mean ± SEM ( n = 3). Immunoblots are representative of n = 3. * P

    Article Snippet: Bafilomycin A1 was purchased from LC Laboratories.

    Techniques: Inhibition, shRNA, Western Blot, Expressing, Infection, Luciferase, Reporter Assay, Construct, Stable Transfection, Transfection

    Inhibition of V-ATPase blocks ABCA1 mediated cholesterol efflux. ABCA1 induced (black bars) and non-induced (open bars) BHK cells were pretreated with 5 nM to 15 nM bafilomycin A1 for 16 hr ( A ), or 10 nM or 10 μM bafilomycin A1 for 1 hr ( B ), and % [ 3 H]cholesterol efflux was determined after a 4 hr chase +/− 5 μg/ml apoA1 (N=3; mean ± SD; groups with different letters above the bars show P ≤0.005 by ANOVA posttest). RAW264.7 cells were transfected with scramble or ATP6V 0 C siRNA by Nucleofection and ATP6V 0 C mRNA levels were measured by qPCR analysis normalized to β-actin mRNA ( C , N=3; mean ± SD; P

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Vacuolar ATPase Activity Required for ABCA1 Mediated Cholesterol Efflux

    doi: 10.1161/ATVBAHA.118.311814

    Figure Lengend Snippet: Inhibition of V-ATPase blocks ABCA1 mediated cholesterol efflux. ABCA1 induced (black bars) and non-induced (open bars) BHK cells were pretreated with 5 nM to 15 nM bafilomycin A1 for 16 hr ( A ), or 10 nM or 10 μM bafilomycin A1 for 1 hr ( B ), and % [ 3 H]cholesterol efflux was determined after a 4 hr chase +/− 5 μg/ml apoA1 (N=3; mean ± SD; groups with different letters above the bars show P ≤0.005 by ANOVA posttest). RAW264.7 cells were transfected with scramble or ATP6V 0 C siRNA by Nucleofection and ATP6V 0 C mRNA levels were measured by qPCR analysis normalized to β-actin mRNA ( C , N=3; mean ± SD; P

    Article Snippet: ABCA1 mediated cholesterol efflux to apoA1 was dose dependently inhibited by 5 to 15 nM bafilomycin A1 with 16 hr pretreatment , and 10 nM to 10 μM bafilomycin A1 with 1 hr pretreatment ( ) in RAW cells.

    Techniques: Inhibition, Transfection, Real-time Polymerase Chain Reaction

    Inhibition V-ATPase by bafilomycin A1 inhibits apoA1 acidification. ABCA1 induced and non-induced BHK cells were pretreated +/− 10 nM ( A ) or 10 μM ( B ) bafilomycin A1 for 1 hr. Cells were then incubated with 1 μg/ml FITC/Alexa647 apoA1 for 45 min at 37 o C +/− bafilomycin A1 followed by flow cytometry to measure the FITC/Alexa647 ratio as indication of apoA1 acidification. Plot shows the frequency histogram of FITC/Alexa647 ratio; representative of 3 independent experiments. C . FITC/Alexa647 labeled apoA1 uptake by BHK cells with or without ABCA1 induction was not inhibited by treatment with 10 μM bafilomycin A1 for 1 hr, as assessed by Alexa647 flow cytometry (N=3, mean ± SD, different letters above the bars show P

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Vacuolar ATPase Activity Required for ABCA1 Mediated Cholesterol Efflux

    doi: 10.1161/ATVBAHA.118.311814

    Figure Lengend Snippet: Inhibition V-ATPase by bafilomycin A1 inhibits apoA1 acidification. ABCA1 induced and non-induced BHK cells were pretreated +/− 10 nM ( A ) or 10 μM ( B ) bafilomycin A1 for 1 hr. Cells were then incubated with 1 μg/ml FITC/Alexa647 apoA1 for 45 min at 37 o C +/− bafilomycin A1 followed by flow cytometry to measure the FITC/Alexa647 ratio as indication of apoA1 acidification. Plot shows the frequency histogram of FITC/Alexa647 ratio; representative of 3 independent experiments. C . FITC/Alexa647 labeled apoA1 uptake by BHK cells with or without ABCA1 induction was not inhibited by treatment with 10 μM bafilomycin A1 for 1 hr, as assessed by Alexa647 flow cytometry (N=3, mean ± SD, different letters above the bars show P

    Article Snippet: ABCA1 mediated cholesterol efflux to apoA1 was dose dependently inhibited by 5 to 15 nM bafilomycin A1 with 16 hr pretreatment , and 10 nM to 10 μM bafilomycin A1 with 1 hr pretreatment ( ) in RAW cells.

    Techniques: Inhibition, Incubation, Flow Cytometry, Cytometry, Labeling

    ABCA1 increases V-ATPase on the cell surface. A. Cell surface proteins from ABCA1 induced and non-induced BHK cells were biotinylated, treated with the cell permeable cross linker DSP, and separated from the cellular lysate by streptavidin pull down. Total and cell surface V-ATPase subunits were detected by Western blot analysis. B . ABCA1 induced and non-induced BHK cells were fixed without permeabilization and were stained with antibody against V-ATPase V 0 A1. Flow cytometry was used to measure cell surface V-ATPase (N=3; mean ± SD; P =0.0004). C . ABCA1 induced and non-induced BHK cells were pretreated +/−10 μM bafilomycin A1 for 1 hr, fixed, permeabilized, and stained with antibody against V-ATPase V 0 A1 followed by epifluorescent microscopy (40× objective) for V-ATPase (red) and DAPI (blue). ABCA1 expression increased the level of V-ATPase on the cell surface (white arrows) independent of bafilomycin A1 treatment.

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Vacuolar ATPase Activity Required for ABCA1 Mediated Cholesterol Efflux

    doi: 10.1161/ATVBAHA.118.311814

    Figure Lengend Snippet: ABCA1 increases V-ATPase on the cell surface. A. Cell surface proteins from ABCA1 induced and non-induced BHK cells were biotinylated, treated with the cell permeable cross linker DSP, and separated from the cellular lysate by streptavidin pull down. Total and cell surface V-ATPase subunits were detected by Western blot analysis. B . ABCA1 induced and non-induced BHK cells were fixed without permeabilization and were stained with antibody against V-ATPase V 0 A1. Flow cytometry was used to measure cell surface V-ATPase (N=3; mean ± SD; P =0.0004). C . ABCA1 induced and non-induced BHK cells were pretreated +/−10 μM bafilomycin A1 for 1 hr, fixed, permeabilized, and stained with antibody against V-ATPase V 0 A1 followed by epifluorescent microscopy (40× objective) for V-ATPase (red) and DAPI (blue). ABCA1 expression increased the level of V-ATPase on the cell surface (white arrows) independent of bafilomycin A1 treatment.

    Article Snippet: ABCA1 mediated cholesterol efflux to apoA1 was dose dependently inhibited by 5 to 15 nM bafilomycin A1 with 16 hr pretreatment , and 10 nM to 10 μM bafilomycin A1 with 1 hr pretreatment ( ) in RAW cells.

    Techniques: Western Blot, Staining, Flow Cytometry, Cytometry, Microscopy, Expressing

    Hyperosmolarity does not influence autophagic flux in NP cells. ( a ) Acridine orange staining of NP cells cultured under iso- (330 mOsm/kg H 2 O, top row) or hyperosmotic (500 mOsm/kg H 2 O, bottom row) condition, treated with (right) or without (left) bafilomycin A1. Hyperosmotic stimulus alone showed no change in number of acidic organelles. Bafilomycin A1 significantly reduced the acridine orange staining irrespective of osmolarity. Scale bar, 35 μm. ( b ) Quantification of acridine orange staining confirmed that hyperosmolarity had no effect on the number of acidic organelles. ( c ) LC3 immunofluorescence staining of NP cells cultured under hyperosmolarity with or without bafilomycin A1 treatment. Hyperosmotic stimulus did not upregulate LC3-positive autophagosomes. Scale bar, 20 μm. ( d ) Western blot analysis of NP cells cultured under increasing osmolarity (330–600 mOsm/kg H 2 O) with or without bafilomycin A1 treatment. The accumulation of LC3-II with bafilomycin A1 treatment was similar under iso- and hyperosmotic conditions. SQSTM1 also showed a trend of accumulation with bafilomycin A1 treatment under all conditions. The levels of ATG12-ATG5 and BECN1 remained unaltered between the experimental groups. ( e – h ) Densitometric analyses of multiple Western blots shown in ( d ). Bars represent mean ± SEM (n = 5). Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. NS, non-significant. BafA1, bafilomycin A1. Western blot images were cropped and acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images.

    Journal: Scientific Reports

    Article Title: Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus

    doi: 10.1038/s41598-017-04876-2

    Figure Lengend Snippet: Hyperosmolarity does not influence autophagic flux in NP cells. ( a ) Acridine orange staining of NP cells cultured under iso- (330 mOsm/kg H 2 O, top row) or hyperosmotic (500 mOsm/kg H 2 O, bottom row) condition, treated with (right) or without (left) bafilomycin A1. Hyperosmotic stimulus alone showed no change in number of acidic organelles. Bafilomycin A1 significantly reduced the acridine orange staining irrespective of osmolarity. Scale bar, 35 μm. ( b ) Quantification of acridine orange staining confirmed that hyperosmolarity had no effect on the number of acidic organelles. ( c ) LC3 immunofluorescence staining of NP cells cultured under hyperosmolarity with or without bafilomycin A1 treatment. Hyperosmotic stimulus did not upregulate LC3-positive autophagosomes. Scale bar, 20 μm. ( d ) Western blot analysis of NP cells cultured under increasing osmolarity (330–600 mOsm/kg H 2 O) with or without bafilomycin A1 treatment. The accumulation of LC3-II with bafilomycin A1 treatment was similar under iso- and hyperosmotic conditions. SQSTM1 also showed a trend of accumulation with bafilomycin A1 treatment under all conditions. The levels of ATG12-ATG5 and BECN1 remained unaltered between the experimental groups. ( e – h ) Densitometric analyses of multiple Western blots shown in ( d ). Bars represent mean ± SEM (n = 5). Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. NS, non-significant. BafA1, bafilomycin A1. Western blot images were cropped and acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images.

    Article Snippet: Acridine Orange Staining NP cells were plated in 24-well plates, and cultured in control (330 mOsm/kg H2 O) or hyperosmotic medium (500 mOsm/kg H2 O) with or without rapamycin or bafilomycin A1.

    Techniques: Staining, Cell Culture, Immunofluorescence, Western Blot

    MCU knockdown has no effect on SV exocytosis. Neurons transfected with syp-pHluorin and either shRNA targeting MCU ( shMcu1 and shMcu2 ) or a scrambled control ( shScr ) were stimulated with an action potential train (10 Hz, 120 s, indicated by the bar ) in the presence of bafilomycin A1. A , average traces for syp-pHluorin fluorescence (ΔF/F 0 ± S.E.) under each condition normalized to maximal fluorescence obtained in alkaline buffer (total SV recycling pool). B , average peak Syp-pHluorin fluorescence normalized to the total SV recycling pool. C, mean SV exocytosis rate calculated using a linear fit to Syp-pHluorin fluorescence during the first 12 s of stimulation. B and C , data are mean ± S.E. (shScr, n = 6; shMcu1, n = 7; shMcu2, n = 5; one-way ANOVA with Holm-Šídák post hoc tests; all non-significant; p > 0.05).

    Journal: The Journal of Biological Chemistry

    Article Title: Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals *

    doi: 10.1074/jbc.M115.686956

    Figure Lengend Snippet: MCU knockdown has no effect on SV exocytosis. Neurons transfected with syp-pHluorin and either shRNA targeting MCU ( shMcu1 and shMcu2 ) or a scrambled control ( shScr ) were stimulated with an action potential train (10 Hz, 120 s, indicated by the bar ) in the presence of bafilomycin A1. A , average traces for syp-pHluorin fluorescence (ΔF/F 0 ± S.E.) under each condition normalized to maximal fluorescence obtained in alkaline buffer (total SV recycling pool). B , average peak Syp-pHluorin fluorescence normalized to the total SV recycling pool. C, mean SV exocytosis rate calculated using a linear fit to Syp-pHluorin fluorescence during the first 12 s of stimulation. B and C , data are mean ± S.E. (shScr, n = 6; shMcu1, n = 7; shMcu2, n = 5; one-way ANOVA with Holm-Šídák post hoc tests; all non-significant; p > 0.05).

    Article Snippet: For experiments examining exocytosis kinetics and SV pool size, the imaging buffer also contained 1 μm bafilomycin A1 to inhibit the acidification of retrieved SVs.

    Techniques: Transfection, shRNA, Fluorescence

    SLC9 PROTAC Series (A) The chemical structure of the SLC degrader d9A-2. See Figure S5 A for structures of the related molecules. (B) HAP1 and KBM7 cells were treated with different concentrations of d9A-2. Within 8 h, degradation of SLC9A1 was observed in both cell lines. (C) Both WT and CRBN knockout KBM7 cell lines were treated with indicated concentrations of d9A-2 for 12 h. SLC9A1 degradation is observed in WT but not CRBN knockout. (D) Chemical “rescue” of d9A-2-driven degradation. WT KBM7 cell lines were treated with 0.5 μM d9A-2 for 16 h. These cells were also treated with bortezomib (bort.) (0.25 μM), MG-132 (MG) (1 μM), MLN4924 (MLN) (1 μM), pomalidomide (poma.) (1 μM), or bafilomycin A1 (bafi.) (10 μM). All molecules, apart from bafilomycin, could rescue SLC9A1 from degradation. (E) Selectivity of d9A-2 was tested across HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SLC9B1, and SLC9B2. Cells were treated with varying concentrations of d9A-2 for 16 h, after which degradation of the exogenous SLCs was monitored. At 0.25 μM, SLC9A1 is the only SLC9 member that is completely degraded. (F) Kinetics of d9A-2-induced degradation tested in HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A4, and SLC9A6. d9A-2 (0.75 μM) was added to HAP1 cell lines for the indicated length of time. SLC9A1 is the only protein that is mostly degraded after 6 h.

    Journal: Cell Chemical Biology

    Article Title: Targeted Degradation of SLC Transporters Reveals Amenability of Multi-Pass Transmembrane Proteins to Ligand-Induced Proteolysis

    doi: 10.1016/j.chembiol.2020.04.003

    Figure Lengend Snippet: SLC9 PROTAC Series (A) The chemical structure of the SLC degrader d9A-2. See Figure S5 A for structures of the related molecules. (B) HAP1 and KBM7 cells were treated with different concentrations of d9A-2. Within 8 h, degradation of SLC9A1 was observed in both cell lines. (C) Both WT and CRBN knockout KBM7 cell lines were treated with indicated concentrations of d9A-2 for 12 h. SLC9A1 degradation is observed in WT but not CRBN knockout. (D) Chemical “rescue” of d9A-2-driven degradation. WT KBM7 cell lines were treated with 0.5 μM d9A-2 for 16 h. These cells were also treated with bortezomib (bort.) (0.25 μM), MG-132 (MG) (1 μM), MLN4924 (MLN) (1 μM), pomalidomide (poma.) (1 μM), or bafilomycin A1 (bafi.) (10 μM). All molecules, apart from bafilomycin, could rescue SLC9A1 from degradation. (E) Selectivity of d9A-2 was tested across HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SLC9B1, and SLC9B2. Cells were treated with varying concentrations of d9A-2 for 16 h, after which degradation of the exogenous SLCs was monitored. At 0.25 μM, SLC9A1 is the only SLC9 member that is completely degraded. (F) Kinetics of d9A-2-induced degradation tested in HAP1 cell lines expressing Strep/HA-SLC9A1, SLC9A2, SLC9A4, and SLC9A6. d9A-2 (0.75 μM) was added to HAP1 cell lines for the indicated length of time. SLC9A1 is the only protein that is mostly degraded after 6 h.

    Article Snippet: Bafilomycin A1 was obtained from Enzo Life Sciences and Chloroquine from Inivogen.

    Techniques: Knock-Out, Expressing

    Characteristics of Targeted Degradation of SLCs by dTAG System (A) A range of dTAG13 concentrations was tested in cell lines expressing dTAG-HA SLC38A2, SLC16A1, or SLC2A1 for 48 h. The dose required for close to complete degradation varies for these example SLCs. Additional examples are in Figure S3 A. (B) A time course of dTAG-driven SLC degradation. HAP1 cell lines expressing dTAG-HA SLC38A2, SLC9A1, or SLC1A5 were treated with 0.5 μM dTAG7 or dTAG13, and samples were harvested at several time points. The glycosylated form of SLC38A2 (upper band) appeared to be degraded slightly faster than the unglycosylated form. SLC9A1 and SLC1A5 provide additional examples of variation in time required for degradation. Additional SLCs are in Figure S2 D. (C) dTAG-HA SLC2A3 was stably expressed in HAP1, LS180, and HCT15 cells. Following 72 h of treatment with 0.5 μM dTAG13, SLC2A3 was completely degraded. (D) Chemical “rescue” of dTAG-driven degradation of SLCs. HAP1 cell lines expressing dTAG-HA SLC1A5 or SLC38A9 were treated with 0.5 μM dTAG7 for 12 or 18 h, respectively. These cells were also treated with chloroquine (CQ) (50 μM), bortezomib (bort.) (1 μM), MG-132 (MG) (1 μM), MLN4924 (MLN) (1 μM), pomalidomide (poma.) (10 μM), or bafilomycin A1 (bafi.) (2.5 μM). SLC degradation was rescued by inhibiting CRL activity or the proteasome, but not by inhibiting the lysosome machinery. See also Figures S3 D and S3E.

    Journal: Cell Chemical Biology

    Article Title: Targeted Degradation of SLC Transporters Reveals Amenability of Multi-Pass Transmembrane Proteins to Ligand-Induced Proteolysis

    doi: 10.1016/j.chembiol.2020.04.003

    Figure Lengend Snippet: Characteristics of Targeted Degradation of SLCs by dTAG System (A) A range of dTAG13 concentrations was tested in cell lines expressing dTAG-HA SLC38A2, SLC16A1, or SLC2A1 for 48 h. The dose required for close to complete degradation varies for these example SLCs. Additional examples are in Figure S3 A. (B) A time course of dTAG-driven SLC degradation. HAP1 cell lines expressing dTAG-HA SLC38A2, SLC9A1, or SLC1A5 were treated with 0.5 μM dTAG7 or dTAG13, and samples were harvested at several time points. The glycosylated form of SLC38A2 (upper band) appeared to be degraded slightly faster than the unglycosylated form. SLC9A1 and SLC1A5 provide additional examples of variation in time required for degradation. Additional SLCs are in Figure S2 D. (C) dTAG-HA SLC2A3 was stably expressed in HAP1, LS180, and HCT15 cells. Following 72 h of treatment with 0.5 μM dTAG13, SLC2A3 was completely degraded. (D) Chemical “rescue” of dTAG-driven degradation of SLCs. HAP1 cell lines expressing dTAG-HA SLC1A5 or SLC38A9 were treated with 0.5 μM dTAG7 for 12 or 18 h, respectively. These cells were also treated with chloroquine (CQ) (50 μM), bortezomib (bort.) (1 μM), MG-132 (MG) (1 μM), MLN4924 (MLN) (1 μM), pomalidomide (poma.) (10 μM), or bafilomycin A1 (bafi.) (2.5 μM). SLC degradation was rescued by inhibiting CRL activity or the proteasome, but not by inhibiting the lysosome machinery. See also Figures S3 D and S3E.

    Article Snippet: Bafilomycin A1 was obtained from Enzo Life Sciences and Chloroquine from Inivogen.

    Techniques: Expressing, Stable Transfection, Activity Assay

    ATP6V0C regulation of autophagy-lysosome pathway markers. Representative western blots for lysosome marker LAMP-1 (A) or autophagosome marker LC3-II (B) from lysates collected following nucleofection with Non-target or ATP6V0C siRNA and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels C–F. Within group comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for LC3-II (C) or LAMP-1 (D) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Journal: PLoS ONE

    Article Title: ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease

    doi: 10.1371/journal.pone.0093257

    Figure Lengend Snippet: ATP6V0C regulation of autophagy-lysosome pathway markers. Representative western blots for lysosome marker LAMP-1 (A) or autophagosome marker LC3-II (B) from lysates collected following nucleofection with Non-target or ATP6V0C siRNA and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels C–F. Within group comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for LC3-II (C) or LAMP-1 (D) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Article Snippet: Fold changes between observed following treatment with 10 nM (1.31±0.24) or 100 nM (1.12±0.16) bafilomycin A1 were not significantly different.

    Techniques: Western Blot, Marker, Concentration Assay, Expressing

    Functional validation of ATP6V0C knockdown. Vesicular acidification following siRNA-mediated knockdown of ATP6V0C was assessed using Lysotracker Red (LTR). LTR-positive punctae were imaged in differentiated SH-SY5Y cells at 96 h following nucleofection of either Non-target siRNA control (A–C) or ATP6V0C siRNA (D–F). DMSO vehicle or 100 nM bafilomycin A1 (BafA1) was added to cells at 3 h prior to the addition of LTR. Scale bar = 20 μm. Inset boxes in panels A and D are magnified in panels B and E respectively, with arrows indicating LTR-positive punctae. (G) Flow cytometric quantification of LTR relative mean fluorescence intensity (MFI) in vehicle-treated cells at 96 h following nucleofection with Non-target vs. ATP6V0C siRNA. Data are expressed as mean ± SEM and are represented by six independent experiments. # p

    Journal: PLoS ONE

    Article Title: ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease

    doi: 10.1371/journal.pone.0093257

    Figure Lengend Snippet: Functional validation of ATP6V0C knockdown. Vesicular acidification following siRNA-mediated knockdown of ATP6V0C was assessed using Lysotracker Red (LTR). LTR-positive punctae were imaged in differentiated SH-SY5Y cells at 96 h following nucleofection of either Non-target siRNA control (A–C) or ATP6V0C siRNA (D–F). DMSO vehicle or 100 nM bafilomycin A1 (BafA1) was added to cells at 3 h prior to the addition of LTR. Scale bar = 20 μm. Inset boxes in panels A and D are magnified in panels B and E respectively, with arrows indicating LTR-positive punctae. (G) Flow cytometric quantification of LTR relative mean fluorescence intensity (MFI) in vehicle-treated cells at 96 h following nucleofection with Non-target vs. ATP6V0C siRNA. Data are expressed as mean ± SEM and are represented by six independent experiments. # p

    Article Snippet: Fold changes between observed following treatment with 10 nM (1.31±0.24) or 100 nM (1.12±0.16) bafilomycin A1 were not significantly different.

    Techniques: Functional Assay, Flow Cytometry, Fluorescence

    ATP6V0C knockdown exhibit enhanced markers of cytotoxicity. Representative confocal microscopy images obtained from differentiated SH-SY5Y cells following nucleofection with Non-target (A–D) or ATP6V0C (E–H) siRNA and subsequent treatment for 48 h with 0 (A, E), 1 (B, F), 3 (C, G) or 10 (D, H) nM bafilomycin A1 (BafA1). Arrows indicate neuritic processes. Scale bar = 50 μm. Neurite length (μm) is expressed graphically (I) and represents results (mean ± SEM) from three independent experiments (10 cells per condition in each experiment). Percent cell death (percentage of propidium iodide (PI)-positive cells quantified using flow cytometry) is expressed graphically (J) as mean ± SEM with data obtained from a total of nine independent experiments. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Journal: PLoS ONE

    Article Title: ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease

    doi: 10.1371/journal.pone.0093257

    Figure Lengend Snippet: ATP6V0C knockdown exhibit enhanced markers of cytotoxicity. Representative confocal microscopy images obtained from differentiated SH-SY5Y cells following nucleofection with Non-target (A–D) or ATP6V0C (E–H) siRNA and subsequent treatment for 48 h with 0 (A, E), 1 (B, F), 3 (C, G) or 10 (D, H) nM bafilomycin A1 (BafA1). Arrows indicate neuritic processes. Scale bar = 50 μm. Neurite length (μm) is expressed graphically (I) and represents results (mean ± SEM) from three independent experiments (10 cells per condition in each experiment). Percent cell death (percentage of propidium iodide (PI)-positive cells quantified using flow cytometry) is expressed graphically (J) as mean ± SEM with data obtained from a total of nine independent experiments. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Article Snippet: Fold changes between observed following treatment with 10 nM (1.31±0.24) or 100 nM (1.12±0.16) bafilomycin A1 were not significantly different.

    Techniques: Confocal Microscopy, Flow Cytometry, Cytometry, Concentration Assay

    ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein. Representative western blot for α-syn (A) from lysates following nucleofection and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1), indicating α-syn high molecular weight (HMW) species ( > 50 kDa, suggesting multimeric species) and α-syn monomer (∼17 kDa). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels B–E. Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for α-syn HMW species (C) or monomer (D) by expressing mean ± SEM band intensities relative to actin loading control (results of one-way ANOVA were not significant, p > 0.05). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for α-syn HMW species (D) or monomer (E) were determined for each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control. # p

    Journal: PLoS ONE

    Article Title: ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease

    doi: 10.1371/journal.pone.0093257

    Figure Lengend Snippet: ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein. Representative western blot for α-syn (A) from lysates following nucleofection and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1), indicating α-syn high molecular weight (HMW) species ( > 50 kDa, suggesting multimeric species) and α-syn monomer (∼17 kDa). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels B–E. Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for α-syn HMW species (C) or monomer (D) by expressing mean ± SEM band intensities relative to actin loading control (results of one-way ANOVA were not significant, p > 0.05). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for α-syn HMW species (D) or monomer (E) were determined for each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control. # p

    Article Snippet: Fold changes between observed following treatment with 10 nM (1.31±0.24) or 100 nM (1.12±0.16) bafilomycin A1 were not significantly different.

    Techniques: Western Blot, Molecular Weight, Concentration Assay, Expressing

    ATP6V0C regulates basal and stress-induced metabolism of APP. Representative western blot (A) for amyloid precursor protein C-terminal fragments (APP CTFs) from nucleofected cells following 48 h treatment with 0–100 nM bafilomycin A1 (BafA1). An antibody was used that recognized both full-length APP (∼110 kDa) and APP CTFs (≤15 kDa). Sizes indicated for CTFs are predicted relative to migration of molecular weight marker and correlate to sizes as previously published (please see results section for further information). In addition to a short-exposure (5 min) blot, a long-exposure (2 h) blot is shown to indicate APP CTFs in vehicle-treated cells. Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. APP CTFs from long-exposure blots quantified from four independent experiments are expressed graphically (B–C). Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined (B) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Journal: PLoS ONE

    Article Title: ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease

    doi: 10.1371/journal.pone.0093257

    Figure Lengend Snippet: ATP6V0C regulates basal and stress-induced metabolism of APP. Representative western blot (A) for amyloid precursor protein C-terminal fragments (APP CTFs) from nucleofected cells following 48 h treatment with 0–100 nM bafilomycin A1 (BafA1). An antibody was used that recognized both full-length APP (∼110 kDa) and APP CTFs (≤15 kDa). Sizes indicated for CTFs are predicted relative to migration of molecular weight marker and correlate to sizes as previously published (please see results section for further information). In addition to a short-exposure (5 min) blot, a long-exposure (2 h) blot is shown to indicate APP CTFs in vehicle-treated cells. Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. APP CTFs from long-exposure blots quantified from four independent experiments are expressed graphically (B–C). Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined (B) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p

    Article Snippet: Fold changes between observed following treatment with 10 nM (1.31±0.24) or 100 nM (1.12±0.16) bafilomycin A1 were not significantly different.

    Techniques: Western Blot, Migration, Molecular Weight, Marker, Concentration Assay, Expressing

    GAS effector Nga inhibits starvation-induced LC3-puncta formation. Confocal micrographs (a) and quantification of starvation-induced LC3-puncta formation (b) and GcAV formation (c and d). HeLa cells stably expressing GFP-LC3 infected with GAS wild-type or its mutants for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Cells were fixed and immunostained for GAC (group A carbohydrate) to observe GAS (red). (e and f) Conversion of LC3-I to LC3-II in response to starvation during GAS infection. HeLa cells were infected with indicated GAS strains for 2 h and incubated with regular (control) or starvation medium for 1 h with or without bafilomycin A1. Representative WB images of LC3 and GAPDH (control) (E) and quantification of LC3 conversion (LC3-I to LC3-II) (f). Intensity ratio of LC3-II:LC3-I were normalized to that in non-infected (NI) cells without bafilomycin A1. (g) Confocal micrographs of starvation-induced LC3-puncta formation. HeLa cells stably expressing GFP-LC3 infected with GAS Nga R289K,G330D for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Scale bars: 10 μm. Data in (b, c, d, and f) are mean ± SEM of 3 independent experiments. Data were tested by two-tailed Student’s t-test: *** P

    Journal: Autophagy

    Article Title: Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

    doi: 10.1080/15548627.2019.1628539

    Figure Lengend Snippet: GAS effector Nga inhibits starvation-induced LC3-puncta formation. Confocal micrographs (a) and quantification of starvation-induced LC3-puncta formation (b) and GcAV formation (c and d). HeLa cells stably expressing GFP-LC3 infected with GAS wild-type or its mutants for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Cells were fixed and immunostained for GAC (group A carbohydrate) to observe GAS (red). (e and f) Conversion of LC3-I to LC3-II in response to starvation during GAS infection. HeLa cells were infected with indicated GAS strains for 2 h and incubated with regular (control) or starvation medium for 1 h with or without bafilomycin A1. Representative WB images of LC3 and GAPDH (control) (E) and quantification of LC3 conversion (LC3-I to LC3-II) (f). Intensity ratio of LC3-II:LC3-I were normalized to that in non-infected (NI) cells without bafilomycin A1. (g) Confocal micrographs of starvation-induced LC3-puncta formation. HeLa cells stably expressing GFP-LC3 infected with GAS Nga R289K,G330D for 2 h, and subsequently incubated with regular (control), starvation medium for 1 h. Scale bars: 10 μm. Data in (b, c, d, and f) are mean ± SEM of 3 independent experiments. Data were tested by two-tailed Student’s t-test: *** P

    Article Snippet: The following reagents were used: Bafilomycin A1 (InvivoGen, tlrl-baf1), wortmannin (Tocris Biaoscience, 1232), rapamycin (Nacalai Tesque, 30,037–94), LysoTracker Red DND-99 (Molecular Probes/Invitrogen Corporation, L7528), puromycin (InvivoGen, ant-pr-1).

    Techniques: Stable Transfection, Expressing, Infection, Incubation, Western Blot, Two Tailed Test

    Autophagy modulates the sensitivity of GBM primary cells to chemotherapy. a The effects of autophagy to chemoresistance were confirmed with primary cells from GBM patients. Similar to what was observed with GBM cell lines, upon 5-day drug treatment, while autophagy inhibition by bafilomycin A1 sensitized the primary cancer cells to death, autophagy induction by rapamycin dramatically attenuated the cytotoxicity of chemotherapy and promoted cancer cell survival. b As determined by the AAV-mRFP-GFP-LC3B reporter assay, for all the groups, most of the surviving cells manifested high level of autophagic activity after 5-day treatment, suggesting that autophagy underlies the chemoresistance of GBM primary cells

    Journal: Cell Death & Disease

    Article Title: Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival

    doi: 10.1038/s41419-017-0242-x

    Figure Lengend Snippet: Autophagy modulates the sensitivity of GBM primary cells to chemotherapy. a The effects of autophagy to chemoresistance were confirmed with primary cells from GBM patients. Similar to what was observed with GBM cell lines, upon 5-day drug treatment, while autophagy inhibition by bafilomycin A1 sensitized the primary cancer cells to death, autophagy induction by rapamycin dramatically attenuated the cytotoxicity of chemotherapy and promoted cancer cell survival. b As determined by the AAV-mRFP-GFP-LC3B reporter assay, for all the groups, most of the surviving cells manifested high level of autophagic activity after 5-day treatment, suggesting that autophagy underlies the chemoresistance of GBM primary cells

    Article Snippet: After overnight incubation, cells were subjected to drug treatment for up to 5 days as indicated under normal or glucose starvation medium: temozolomide (200 μM, M2129, Abmole), carboplatin (50 μM, M2288, Abmole), respectively, with or without autophagy inhibitors/agonist, bafilomycin A1 (10 nM, A601116, Sangon), 3-methyladenine (2 mM, S2767, Selleck), hydroxychloroquine (100 nM, S4430, Selleck), MHY1485 (25 μM, S7811, Selleck) and rapamycin (5 μM, S1039, Selleck).

    Techniques: Inhibition, Reporter Assay, Activity Assay

    Lipid loading does not affect macrophage autophagy: ex vivo and in vitro studies A. Neutral lipid species in total eWAT ATMs of LFD and HFD mice were detected by LipidTox ™ staining. A representative image is shown. B. Quantification of neutral lipid species represented in A, n=15 mice/group. C. Neutral lipid species in BMDMs treated for 24 h with 500 μM palmitate and 500 μM oleate were detected by LipidTox ™ staining. A representative image is shown. D. Quantification of neutral lipid species represented in C, BMDMs from n=4 mice/group. E – J. The ratio of LC3-II:LC3-I or levels of p62 normalized to tubulin is reported by Western blotting in BMDMs treated with the indicated fatty acid for 24 h from n=4 mice/group. Where indicated, BMDMs were treated with 10 μm bafilomycin for the last 3 h in the 24 h fatty acid treatment time period. F and G are quantifications of E. I and J and quantifications of H. FMO = fluorescence minus one, Palm = palmitate, Baf = bafilomycin * = p

    Journal: Obesity research & clinical practice

    Article Title: Myeloid ATG16L1 does not affect adipose tissue inflammation or body mass in mice fed high fat diet

    doi: 10.1016/j.orcp.2017.10.006

    Figure Lengend Snippet: Lipid loading does not affect macrophage autophagy: ex vivo and in vitro studies A. Neutral lipid species in total eWAT ATMs of LFD and HFD mice were detected by LipidTox ™ staining. A representative image is shown. B. Quantification of neutral lipid species represented in A, n=15 mice/group. C. Neutral lipid species in BMDMs treated for 24 h with 500 μM palmitate and 500 μM oleate were detected by LipidTox ™ staining. A representative image is shown. D. Quantification of neutral lipid species represented in C, BMDMs from n=4 mice/group. E – J. The ratio of LC3-II:LC3-I or levels of p62 normalized to tubulin is reported by Western blotting in BMDMs treated with the indicated fatty acid for 24 h from n=4 mice/group. Where indicated, BMDMs were treated with 10 μm bafilomycin for the last 3 h in the 24 h fatty acid treatment time period. F and G are quantifications of E. I and J and quantifications of H. FMO = fluorescence minus one, Palm = palmitate, Baf = bafilomycin * = p

    Article Snippet: OptiMem was replaced with DMEM containing palmitate (100 μM) (Sigma, P9767) vs dPBS control or a mixture of palmitate and oleate (Sigma, 07501) (500 μM each) vs fatty acid free BSA (0.17mM) (Roche, 03117057001) conjugate control, 5mM glucose, and 1% P/S for 24 h. 10 μM Bafilomycin (Sigma, B1793) was added to a portion of the palmitate (100 μM) vs dPBS treated cells for the last 3 h of treatment.

    Techniques: Ex Vivo, In Vitro, Mouse Assay, Staining, Western Blot, Fluorescence

    N -Acetyl cysteine reverts the effect of RPIA knockdown on LC3 processing. A) Immunoblot of LC3-processing and S6 Kinase signaling pathway in 293 cells transfected with pLKO.1 control and shRPIA (sh1–3) for 72 h, treated with EBSS, 10 nM bafilomycin A, 250 nM torin 1, 10 nM bafilomycin A + 250 nM torin1 and 1 mM N -acetyl cysteine for 3 h before harvesting. NT – not treated, EBSS - Earle's Balanced Salt Solution, Baf – bafilomycin, Tor – torin 1, and N-Ac – N -acetyl cysteine. B) Luciferase activity of supernatant collected 48 h post-transfection of control (pMOWS) and shRPIA, in 293 T cells expressing Act-LC3-dNGLuc. Cells were treated overnight with N -acetyl cysteine (10 mM). Data represent mean ± SD, n = 3.

    Journal: Cellular Signalling

    Article Title: Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy

    doi: 10.1016/j.cellsig.2016.06.015

    Figure Lengend Snippet: N -Acetyl cysteine reverts the effect of RPIA knockdown on LC3 processing. A) Immunoblot of LC3-processing and S6 Kinase signaling pathway in 293 cells transfected with pLKO.1 control and shRPIA (sh1–3) for 72 h, treated with EBSS, 10 nM bafilomycin A, 250 nM torin 1, 10 nM bafilomycin A + 250 nM torin1 and 1 mM N -acetyl cysteine for 3 h before harvesting. NT – not treated, EBSS - Earle's Balanced Salt Solution, Baf – bafilomycin, Tor – torin 1, and N-Ac – N -acetyl cysteine. B) Luciferase activity of supernatant collected 48 h post-transfection of control (pMOWS) and shRPIA, in 293 T cells expressing Act-LC3-dNGLuc. Cells were treated overnight with N -acetyl cysteine (10 mM). Data represent mean ± SD, n = 3.

    Article Snippet: Following compounds/reagents were used as indicated: bafilomycin A (Sigma®, B1793), puromycin dihydrochloride (Sigma®, P9620), DMSO (Sigma®, D2650) Earle's Balanced Salt Solution (EBSS; ThermoFisher Scientific®, 24010-043), torin 1 (Merck-Millipore, 475991) and N -acetyl cysteine (Sigma, A9165).

    Techniques: Transfection, Luciferase, Activity Assay, Expressing, Activated Clotting Time Assay

    LC3-II levels and LC3 puncta are increased in CRISPR cell lines. A) Immunoblot of LC3-processing in control cells (CR-WT), CR-1 and CR-2, treated with 10 nM bafilomycin A (baf) for 2 h. B) Densitometry analysis of relative LC-II/Actin levels using Fiji. Data show the mean ± SD, n = 5. *, p

    Journal: Cellular Signalling

    Article Title: Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy

    doi: 10.1016/j.cellsig.2016.06.015

    Figure Lengend Snippet: LC3-II levels and LC3 puncta are increased in CRISPR cell lines. A) Immunoblot of LC3-processing in control cells (CR-WT), CR-1 and CR-2, treated with 10 nM bafilomycin A (baf) for 2 h. B) Densitometry analysis of relative LC-II/Actin levels using Fiji. Data show the mean ± SD, n = 5. *, p

    Article Snippet: Following compounds/reagents were used as indicated: bafilomycin A (Sigma®, B1793), puromycin dihydrochloride (Sigma®, P9620), DMSO (Sigma®, D2650) Earle's Balanced Salt Solution (EBSS; ThermoFisher Scientific®, 24010-043), torin 1 (Merck-Millipore, 475991) and N -acetyl cysteine (Sigma, A9165).

    Techniques: CRISPR

    GFP-LC3 puncta are increased upon knockdown of RPIA. A) GFP-LC3 puncta in stably expressing GFP-LC3 cells, transfected with control (pMOWS) and pMOWS-shRPIA, imaged after 96 h knockdown. Cells were treated 1% DMSO or with 10 nM bafilomycin A (baf) in EBSS medium for two hours. Images were acquired on an inverted confocal Leica SPE microscope with a 63 × objective, scale bar = 10 μm. B) Puncta were identified and analyzed using Fiji image analysis software. Data represent mean ± SD of 1000–3000 cells from 2 independent experiments. ***p

    Journal: Cellular Signalling

    Article Title: Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy

    doi: 10.1016/j.cellsig.2016.06.015

    Figure Lengend Snippet: GFP-LC3 puncta are increased upon knockdown of RPIA. A) GFP-LC3 puncta in stably expressing GFP-LC3 cells, transfected with control (pMOWS) and pMOWS-shRPIA, imaged after 96 h knockdown. Cells were treated 1% DMSO or with 10 nM bafilomycin A (baf) in EBSS medium for two hours. Images were acquired on an inverted confocal Leica SPE microscope with a 63 × objective, scale bar = 10 μm. B) Puncta were identified and analyzed using Fiji image analysis software. Data represent mean ± SD of 1000–3000 cells from 2 independent experiments. ***p

    Article Snippet: Following compounds/reagents were used as indicated: bafilomycin A (Sigma®, B1793), puromycin dihydrochloride (Sigma®, P9620), DMSO (Sigma®, D2650) Earle's Balanced Salt Solution (EBSS; ThermoFisher Scientific®, 24010-043), torin 1 (Merck-Millipore, 475991) and N -acetyl cysteine (Sigma, A9165).

    Techniques: Stable Transfection, Expressing, Transfection, Microscopy, Software

    Lysosomal cysteine proteases activate PEDV pseudovirus entry. A and B , Huh-7 cells ( A ) or PK-15 cells ( B ) were preincubated with bafilomycin A1 ( Baf-A1 , a lysosomal acidification inhibitor) or E-64d (a lysosomal cysteine protease inhibitor) at the indicated concentrations and then transduced by PEDV pseudoviruses. C and D , retroviruses pseudotyped with VSV envelop glycoprotein ( i.e. VSV pseudoviruses) were used as a control. The pseudovirus entry in target cells without any inhibitor treatment was taken as 100%. Error bars indicate S.E. ( n = 5).

    Journal: The Journal of Biological Chemistry

    Article Title: Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases *

    doi: 10.1074/jbc.M116.740746

    Figure Lengend Snippet: Lysosomal cysteine proteases activate PEDV pseudovirus entry. A and B , Huh-7 cells ( A ) or PK-15 cells ( B ) were preincubated with bafilomycin A1 ( Baf-A1 , a lysosomal acidification inhibitor) or E-64d (a lysosomal cysteine protease inhibitor) at the indicated concentrations and then transduced by PEDV pseudoviruses. C and D , retroviruses pseudotyped with VSV envelop glycoprotein ( i.e. VSV pseudoviruses) were used as a control. The pseudovirus entry in target cells without any inhibitor treatment was taken as 100%. Error bars indicate S.E. ( n = 5).

    Article Snippet: Briefly, target cells were preincubated with 10 or 50 μ m proprotein convertase inhibitor Dec-RVKR-CMK (Enzo Life Sciences), 20 or 100 μ m camostat mesylate (Sigma-Aldrich), 20 or 100 n m bafilomycin A1 (Sigma-Aldrich), 10 or 50 μ m E-64d (Sigma-Aldrich), 50 μ m cathepsin L inhibitor Z-FY-CHO (Santa Cruz Biotechnology), or 50 μ m cathepsin B inhibitor CA-074 Me (Santa Cruz Biotechnology) at 37 °C for 1 h. The cells were subsequently transduced by retroviruses pseudotyped with PEDV spike, MERS-CoV spike, or VSV envelope glycoprotein.

    Techniques: Protease Inhibitor

    High glucose (HG) stimulates autophagic activity with downstream suppression of autophagic flux in vitro . Autophagic flux in proximal tubular epithelial cells (PTECs) during normal and HG treatment was estimated by the conversion from microtubule-associated protein 1 light chain 3-I (MAP1LC3-I) to MAP1LC3-II as a readout of autophagosome formation ( n =4–5, respectively). (A) Autophagic flux index (defined as the proportion of the levels of MAP1LC3-II in the presence of bafilomycin A1 (BafA1) to the levels of MAP1LC3-II in the absence of BafA1) is calculated at the indicated times. (B) Effect of insulin on autophagic flux in PTECs during HG exposure was assessed. Representative immunoblots are shown. Data are presented as means±SEM. Statistically significant differences are indicated. ACTB, actin, beta; LG, low glucose. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Proximal Tubule Autophagy Differs in Type 1 and 2 Diabetes

    doi: 10.1681/ASN.2018100983

    Figure Lengend Snippet: High glucose (HG) stimulates autophagic activity with downstream suppression of autophagic flux in vitro . Autophagic flux in proximal tubular epithelial cells (PTECs) during normal and HG treatment was estimated by the conversion from microtubule-associated protein 1 light chain 3-I (MAP1LC3-I) to MAP1LC3-II as a readout of autophagosome formation ( n =4–5, respectively). (A) Autophagic flux index (defined as the proportion of the levels of MAP1LC3-II in the presence of bafilomycin A1 (BafA1) to the levels of MAP1LC3-II in the absence of BafA1) is calculated at the indicated times. (B) Effect of insulin on autophagic flux in PTECs during HG exposure was assessed. Representative immunoblots are shown. Data are presented as means±SEM. Statistically significant differences are indicated. ACTB, actin, beta; LG, low glucose. * P

    Article Snippet: To assess autophagic flux, PTECs incubated with low- or high-glucose DMEM for up to 24 hours were treated with 200 nM bafilomycin A1 (BafA1; Wako) for 1 hour before harvest.

    Techniques: Activity Assay, In Vitro, Western Blot

    Chemical Inhibition of VCP ATPase Activity with ML240 Impairs IFITM3 Turnover, Lysosomal Sorting, and Trafficking (A) Analysis of IFITM3 and ubiquitination levels upon treatment with ML240. HEK293T cells were transfected with HA-IFITM3 for 12 h, treated with DMSO or ML240 (2.5 μM) for another 12 h, and then lysed for western blot analysis. The red asterisks indicate IFITM3 ubiquitination bands with high molecular weights. (B) Analysis of IFITM3 turnover upon treatment with ML240. HeLa cells expressing HA-IFITM3 were treated with CHX (25 μg/mL) for the indicated times in the presence of DMSO or ML240 (2.5 μM) and lysed for western blot analysis. (C) Quantification of IFITM3 levels normalized to tubulin levels shown in (B). Data are represented as mean ± SD, n = 3. (D) Immunofluorescence analysis of HA-IFITM3 in the presence of bafilomycin A1 or ML240. HeLa cells were transfected with HA-IFITM3 and mCherry-LAMP1, treated with DMSO, bafilomycin A1 (100 ng/mL), or ML240 (2.5 μM) for 12 h, and processed for immunofluorescence with an Alexa Fluor 488-conjugated anti-HA antibody. Scale bars, 10 μm. The right panels show magnified squared regions in the corresponding left panels. White arrows indicate enlarged IFITM3-containing compartments. (E) Relative percentage of DiD-IAV particles colocalized with IFITM3 at the time of dequenching upon ML240 treatment. HeLa IFITM2/3-KO cells expressing BODIPY-labeled IFITM3 were infected with DiD-IAV particles, acutely treated with ML240 (1 μM), and monitored for DiD dequenching and IFITM3 trafficking by time-lapse imaging. Data are represented as mean ± SD of three independent experiments. ∗∗∗∗p

    Journal: Cell Chemical Biology

    Article Title: Site-Specific Photo-Crosslinking Proteomics Reveal Regulation of IFITM3 Trafficking and Turnover by VCP/p97 ATPase

    doi: 10.1016/j.chembiol.2020.03.004

    Figure Lengend Snippet: Chemical Inhibition of VCP ATPase Activity with ML240 Impairs IFITM3 Turnover, Lysosomal Sorting, and Trafficking (A) Analysis of IFITM3 and ubiquitination levels upon treatment with ML240. HEK293T cells were transfected with HA-IFITM3 for 12 h, treated with DMSO or ML240 (2.5 μM) for another 12 h, and then lysed for western blot analysis. The red asterisks indicate IFITM3 ubiquitination bands with high molecular weights. (B) Analysis of IFITM3 turnover upon treatment with ML240. HeLa cells expressing HA-IFITM3 were treated with CHX (25 μg/mL) for the indicated times in the presence of DMSO or ML240 (2.5 μM) and lysed for western blot analysis. (C) Quantification of IFITM3 levels normalized to tubulin levels shown in (B). Data are represented as mean ± SD, n = 3. (D) Immunofluorescence analysis of HA-IFITM3 in the presence of bafilomycin A1 or ML240. HeLa cells were transfected with HA-IFITM3 and mCherry-LAMP1, treated with DMSO, bafilomycin A1 (100 ng/mL), or ML240 (2.5 μM) for 12 h, and processed for immunofluorescence with an Alexa Fluor 488-conjugated anti-HA antibody. Scale bars, 10 μm. The right panels show magnified squared regions in the corresponding left panels. White arrows indicate enlarged IFITM3-containing compartments. (E) Relative percentage of DiD-IAV particles colocalized with IFITM3 at the time of dequenching upon ML240 treatment. HeLa IFITM2/3-KO cells expressing BODIPY-labeled IFITM3 were infected with DiD-IAV particles, acutely treated with ML240 (1 μM), and monitored for DiD dequenching and IFITM3 trafficking by time-lapse imaging. Data are represented as mean ± SD of three independent experiments. ∗∗∗∗p

    Article Snippet: For pharmacological treatments, Bafilomycin A1 (ab120497) was from Abcam.

    Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Expressing, Immunofluorescence, Labeling, Infection, Imaging

    Detection of autophagy-related markers. (A) Western blot analysis of p62 and LC3-II in DLD-1 scr and DLD-1 KOAGR2 cells in response to treatments as indicated. (B) Changes in the LC3-II level following co-treatment with bafilomycin A1. (C) Western blot

    Journal: Oncology Letters

    Article Title: AGR2 silencing contributes to metformin-dependent sensitization of colorectal cancer cells to chemotherapy

    doi: 10.3892/ol.2019.10800

    Figure Lengend Snippet: Detection of autophagy-related markers. (A) Western blot analysis of p62 and LC3-II in DLD-1 scr and DLD-1 KOAGR2 cells in response to treatments as indicated. (B) Changes in the LC3-II level following co-treatment with bafilomycin A1. (C) Western blot

    Article Snippet: Therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors e.g. bafilomycin A1.

    Techniques: Western Blot

    Increased NAA pathway activity elevated the number of lysosomes and induced autophagy. Mature control and Nat8l o/e iBACs were used for the analysis. (A) Immunoblot of nuclear fraction showing transcription factor EB (TFEB) translocation to the nuclei. Histone H3 was used as loading control for nucleus fraction ( n = 3). (B) Representative electron micrographs of control and Nat8l o/e iBACs and (C) corresponding number of lysosomes, counted from all 90 electron micrographs from 3 biological replicates. Scale bar = 1 μm. L: lysosome, LD: lipid droplet, M: mitochondria N: nucleus. (D) Representative confocal image of iBACs stained with LysoTracker Red ( n = 3). (E) Number of Lysotracker Red stained lysosomes counted by flow cytometry ( n = 3). (F) Autophagic flux was monitored by immunoblotting against LC3 in the presence or absence of autophagy inhibitor bafilomycin A1 (BafA1) GAPDH was used as loading control ( n = 3). (G) Representative confocal images of cells transfected with tandem mRFP-GFP-LC3 vector. GFP and mRFP channels are shown in green and red, respectively. Elevated number of autolysosomes (red puncta in merged image) indicates increased autophagic flux. (H) Immunoblot showing LC3 in cell lysates with or without acetate supplementation to growth media (48 h, 10 mM NaAc) ( n = 3). (I) Densitometry of LC3-II bands from (I) normalized to ACTb ( n = 3). Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t -test (* p

    Journal: Biochimica et biophysica acta. Molecular cell research

    Article Title: N-acetylaspartate pathway is nutrient responsive and coordinates lipid and energy metabolism in brown adipocytes

    doi: 10.1016/j.bbamcr.2018.08.017

    Figure Lengend Snippet: Increased NAA pathway activity elevated the number of lysosomes and induced autophagy. Mature control and Nat8l o/e iBACs were used for the analysis. (A) Immunoblot of nuclear fraction showing transcription factor EB (TFEB) translocation to the nuclei. Histone H3 was used as loading control for nucleus fraction ( n = 3). (B) Representative electron micrographs of control and Nat8l o/e iBACs and (C) corresponding number of lysosomes, counted from all 90 electron micrographs from 3 biological replicates. Scale bar = 1 μm. L: lysosome, LD: lipid droplet, M: mitochondria N: nucleus. (D) Representative confocal image of iBACs stained with LysoTracker Red ( n = 3). (E) Number of Lysotracker Red stained lysosomes counted by flow cytometry ( n = 3). (F) Autophagic flux was monitored by immunoblotting against LC3 in the presence or absence of autophagy inhibitor bafilomycin A1 (BafA1) GAPDH was used as loading control ( n = 3). (G) Representative confocal images of cells transfected with tandem mRFP-GFP-LC3 vector. GFP and mRFP channels are shown in green and red, respectively. Elevated number of autolysosomes (red puncta in merged image) indicates increased autophagic flux. (H) Immunoblot showing LC3 in cell lysates with or without acetate supplementation to growth media (48 h, 10 mM NaAc) ( n = 3). (I) Densitometry of LC3-II bands from (I) normalized to ACTb ( n = 3). Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t -test (* p

    Article Snippet: To investigate autophagy flux by immunoblotting, fully differentiated brown adipocytes were cultivated in maintenance medium supplemented with bafilomycin A1 (100 nM 6 h, Merck, Darmstadt, Germany).

    Techniques: Activity Assay, Translocation Assay, Staining, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Two Tailed Test

    Impact of Nat8l knockdown on energy metabolism, lysosomal biogenesis and autophagy. Mature iBACs with stable knock-down of Nat8l (Nat8l-KD) or control cells were used for the analysis. (A) Analysis of Nat8l , Aspa , and Acss2 mRNA expression ( n = 3). (B) Asp-NAT, ASPA and ACSS2 protein expression ( n = 3). (C–D) NAA and aspartate concentration in iBACs analyzed with HPLC/HRMS ( n = 3). (E) TCA cycle metabolites measured by HPLC/HRMS ( n = 3). (F) Fold change of BCAA measured by NMR ( n = 3). (G) Fold change of amino acids measured by HPLC/HRMS. Significantly reduced and increased AA are depicted in yellow and red, respectively ( n = 3). (H) Number of Lysotracker Red stained lysosomes counted by flow cytometry ( n = 3). (I) Autophagic flux was monitored by immunoblotting against LC3 in presence or absence of inhibitor bafilomycin A1 (BafA1) ( n = 3). (J) Densitometry of LC3-II bands from (I) normalized to ACTb ( n = 3). Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t -test (* p

    Journal: Biochimica et biophysica acta. Molecular cell research

    Article Title: N-acetylaspartate pathway is nutrient responsive and coordinates lipid and energy metabolism in brown adipocytes

    doi: 10.1016/j.bbamcr.2018.08.017

    Figure Lengend Snippet: Impact of Nat8l knockdown on energy metabolism, lysosomal biogenesis and autophagy. Mature iBACs with stable knock-down of Nat8l (Nat8l-KD) or control cells were used for the analysis. (A) Analysis of Nat8l , Aspa , and Acss2 mRNA expression ( n = 3). (B) Asp-NAT, ASPA and ACSS2 protein expression ( n = 3). (C–D) NAA and aspartate concentration in iBACs analyzed with HPLC/HRMS ( n = 3). (E) TCA cycle metabolites measured by HPLC/HRMS ( n = 3). (F) Fold change of BCAA measured by NMR ( n = 3). (G) Fold change of amino acids measured by HPLC/HRMS. Significantly reduced and increased AA are depicted in yellow and red, respectively ( n = 3). (H) Number of Lysotracker Red stained lysosomes counted by flow cytometry ( n = 3). (I) Autophagic flux was monitored by immunoblotting against LC3 in presence or absence of inhibitor bafilomycin A1 (BafA1) ( n = 3). (J) Densitometry of LC3-II bands from (I) normalized to ACTb ( n = 3). Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t -test (* p

    Article Snippet: To investigate autophagy flux by immunoblotting, fully differentiated brown adipocytes were cultivated in maintenance medium supplemented with bafilomycin A1 (100 nM 6 h, Merck, Darmstadt, Germany).

    Techniques: Expressing, Concentration Assay, High Performance Liquid Chromatography, Nuclear Magnetic Resonance, Staining, Flow Cytometry, Cytometry, Two Tailed Test

    Degradation profiles of Golgi-localized model QC substrates. (A) Domain outline of Golgi QC substrates targeted to the cis- (MAN2A1-EGFP-HT2) and trans- (ST6GAL1- and B4GT-EGFP-HT2) Golgi. (B) Flow cytometry analysis quantifying the levels of MAN2A1-EGFP-HT2 in HEK293 cells on the indicated treatments for 6 h (EPX, epoxomicin; BafA1, bafilomycin A). CHX was added 1 h prior to the indicated treatments. (C) Same analysis as in B for HEK293 cells expressing B4GT-EGFP-HT2 or ST6GAL1-EGFP-HT2 ( n = 2, data represent mean ± SEM). (D) Flow cytometry analysis quantifying the levels of B4GT-EGFP-HT2 in HeLa cells on the indicated treatments for 6 h in the absence (left) or presence (right) of CHX ( n = 3, data represent mean ± SEM, results of t test are shown).

    Journal: Molecular Biology of the Cell

    Article Title: Protein folding state-dependent sorting at the Golgi apparatus

    doi: 10.1091/mbc.E19-01-0069

    Figure Lengend Snippet: Degradation profiles of Golgi-localized model QC substrates. (A) Domain outline of Golgi QC substrates targeted to the cis- (MAN2A1-EGFP-HT2) and trans- (ST6GAL1- and B4GT-EGFP-HT2) Golgi. (B) Flow cytometry analysis quantifying the levels of MAN2A1-EGFP-HT2 in HEK293 cells on the indicated treatments for 6 h (EPX, epoxomicin; BafA1, bafilomycin A). CHX was added 1 h prior to the indicated treatments. (C) Same analysis as in B for HEK293 cells expressing B4GT-EGFP-HT2 or ST6GAL1-EGFP-HT2 ( n = 2, data represent mean ± SEM). (D) Flow cytometry analysis quantifying the levels of B4GT-EGFP-HT2 in HeLa cells on the indicated treatments for 6 h in the absence (left) or presence (right) of CHX ( n = 3, data represent mean ± SEM, results of t test are shown).

    Article Snippet: Reagents and antibodies Reagents and antibodies were purchased from the following suppliers: anti-Giantin (Abcam; ab24586, 1:1000), anti-BiP for immunofluorescence (Abcam ab21685, 1:500), anti-BiP for Western blot (Cell signaling, 3183S, 1:1000), anti-KDEL (Abcam ab176333, 1:1000), anti-GFP (Santa Cruz sc-9996, 1:1000), anti-HA (Cell signaling 3724, 1:1000), anti-GAPDH (Cell signaling 2118, 1:2000), TMP (Sigma), CHX (Sigma), doxycycline (Sigma), fibronectin (EMD Millipore), Hoechst (Thermo Fisher Scientific; 10 mg/ml in H2 O, 1:2000) D/D solubilizer (Clonetech), BafA1 (Alfa Aesar), epoxomicin (EMD Millipore), SNAP-tag ligands (SNAP Cell TMR Star and Oregon Green [New England Biolabs]), HT TMR ligand (Promega), and DSP Crosslinker (Thermo Fisher Scientific).

    Techniques: Flow Cytometry, Cytometry, Expressing

    The 7B2-proPC2 complex is not accumulated in the ER but rapidly transported to the Golgi. AtT-20 cells expressing PC2 and 7B2 were labeled for 10 min and chased for the indicated times in the presence of 1 μM bafilomycin A1. PC2 and 7B2 were immunoprecipitated under native conditions, denatured, and digested with endoglycosidase H.

    Journal: The Journal of Cell Biology

    Article Title: Mechanism of the Facilitation of PC2 Maturation by 7B2: Involvement in ProPC2 Transport and Activation but Not Folding

    doi:

    Figure Lengend Snippet: The 7B2-proPC2 complex is not accumulated in the ER but rapidly transported to the Golgi. AtT-20 cells expressing PC2 and 7B2 were labeled for 10 min and chased for the indicated times in the presence of 1 μM bafilomycin A1. PC2 and 7B2 were immunoprecipitated under native conditions, denatured, and digested with endoglycosidase H.

    Article Snippet: Bafilomycin A1 had no effect on the binding of 7B2 to proPC2 (Fig. b ).

    Techniques: Expressing, Labeling, Immunoprecipitation

    7B2 increases proPC2 transport to the Golgi. AtT-20 cells expressing PC2 alone or PC2 and 7B2 were labeled for 10 min and chased for the indicated time periods in the presence of 1 μM bafilomycin A1. PC2 was immunoprecipitated, denatured, and digested with endoglycosidase H. r indicates the position of endoglycosidase H–resistant proPC2, and s the position of endoglycosidase H–sensitive proPC2.

    Journal: The Journal of Cell Biology

    Article Title: Mechanism of the Facilitation of PC2 Maturation by 7B2: Involvement in ProPC2 Transport and Activation but Not Folding

    doi:

    Figure Lengend Snippet: 7B2 increases proPC2 transport to the Golgi. AtT-20 cells expressing PC2 alone or PC2 and 7B2 were labeled for 10 min and chased for the indicated time periods in the presence of 1 μM bafilomycin A1. PC2 was immunoprecipitated, denatured, and digested with endoglycosidase H. r indicates the position of endoglycosidase H–resistant proPC2, and s the position of endoglycosidase H–sensitive proPC2.

    Article Snippet: Bafilomycin A1 had no effect on the binding of 7B2 to proPC2 (Fig. b ).

    Techniques: Expressing, Labeling, Immunoprecipitation

    Bafilomycin A1 effect on proPC2 and 7B2 maturation and binding. ( a ) Bafilomycin A1 prevents the removal of the proPC2 propeptide. AtT-20 cells that express proPC2 alone or proPC2 and 7B2 were labeled for 10 min and directly extracted or chased for 120 min before extraction in the presence or absence of 1 μM bafilomycin A1. PC2 was immunoprecipitated. ( b ) Bafilomycin A1 does not affect 7B2 cleavage and binding to proPC2. AtT-20 cells that express 7B2 and PC2 were labeled for 10 min and directly extracted or chased for 30 min before extraction. 7B2 was immunoprecipitated under native conditions. ( c ) Bafilomycin A1 does not inhibit transport to the Golgi. AtT-20 cells that express 7B2 and PC2 were labeled for 10 min and chased for 120 min before extraction. PC2 was immunoprecipitated, denatured, and digested under control conditions ( C ) or with endoglycosidase H ( H ) or N -glycanase F ( F ).

    Journal: The Journal of Cell Biology

    Article Title: Mechanism of the Facilitation of PC2 Maturation by 7B2: Involvement in ProPC2 Transport and Activation but Not Folding

    doi:

    Figure Lengend Snippet: Bafilomycin A1 effect on proPC2 and 7B2 maturation and binding. ( a ) Bafilomycin A1 prevents the removal of the proPC2 propeptide. AtT-20 cells that express proPC2 alone or proPC2 and 7B2 were labeled for 10 min and directly extracted or chased for 120 min before extraction in the presence or absence of 1 μM bafilomycin A1. PC2 was immunoprecipitated. ( b ) Bafilomycin A1 does not affect 7B2 cleavage and binding to proPC2. AtT-20 cells that express 7B2 and PC2 were labeled for 10 min and directly extracted or chased for 30 min before extraction. 7B2 was immunoprecipitated under native conditions. ( c ) Bafilomycin A1 does not inhibit transport to the Golgi. AtT-20 cells that express 7B2 and PC2 were labeled for 10 min and chased for 120 min before extraction. PC2 was immunoprecipitated, denatured, and digested under control conditions ( C ) or with endoglycosidase H ( H ) or N -glycanase F ( F ).

    Article Snippet: Bafilomycin A1 had no effect on the binding of 7B2 to proPC2 (Fig. b ).

    Techniques: Binding Assay, Labeling, Immunoprecipitation

    Acidic pH causes a modest increase in RNAIII expression in vitro . (A) Phagosomal pH was evaluated in THP-1 macrophages treated with or without the phagosomal acidification inhibitor bafilomycin A1 (Baf; 100 nM) or NH 4 Cl (40 mM) and infected with Oregon

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

    doi: 10.1128/IAI.00704-15

    Figure Lengend Snippet: Acidic pH causes a modest increase in RNAIII expression in vitro . (A) Phagosomal pH was evaluated in THP-1 macrophages treated with or without the phagosomal acidification inhibitor bafilomycin A1 (Baf; 100 nM) or NH 4 Cl (40 mM) and infected with Oregon

    Article Snippet: Briefly, THP-1 cells were treated with 100 nM bafilomycin A1 or DMSO for 1 h and infected with USA300.

    Techniques: Expressing, In Vitro, Infection

    Bafilomycin treatment decreases RNAIII expression in USA300. (A) THP-1 macrophages were treated with bafilomycin A1 (100 nM) or DMSO (vehicle control) and infected with USA300 (MOI of 10). At 1, 2, 4, and 8 h p.i., bacterial RNA was harvested from infected

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

    doi: 10.1128/IAI.00704-15

    Figure Lengend Snippet: Bafilomycin treatment decreases RNAIII expression in USA300. (A) THP-1 macrophages were treated with bafilomycin A1 (100 nM) or DMSO (vehicle control) and infected with USA300 (MOI of 10). At 1, 2, 4, and 8 h p.i., bacterial RNA was harvested from infected

    Article Snippet: Briefly, THP-1 cells were treated with 100 nM bafilomycin A1 or DMSO for 1 h and infected with USA300.

    Techniques: Expressing, Infection

    Inhibitors of phagosomal acidification impair USA300 intracellular survival. THP-1 macrophages were treated with bafilomycin A1 (Baf; 100 nM) or DMSO (vehicle control) for 1 h and then infected with S. aureus strains or S. carnosus (MOI of 10) (A) or

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

    doi: 10.1128/IAI.00704-15

    Figure Lengend Snippet: Inhibitors of phagosomal acidification impair USA300 intracellular survival. THP-1 macrophages were treated with bafilomycin A1 (Baf; 100 nM) or DMSO (vehicle control) for 1 h and then infected with S. aureus strains or S. carnosus (MOI of 10) (A) or

    Article Snippet: Briefly, THP-1 cells were treated with 100 nM bafilomycin A1 or DMSO for 1 h and infected with USA300.

    Techniques: Infection

    Validation of GABARAPL2 endoHA /ACSL3 endoNeonGreen cells. A, GABARAPL2 endoHA and GABARAPL2 endoHA /ACSL3 endoNeonGreen cells as well as parental HeLa cells transiently transfected with TOMM20-NeonGreen were lysed and analyzed by immunoblotting with indicated antibodies. B, Fixed GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were immunostained with an anti-calnexin antibody. Scale bar: 10 µm. C, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were treated with oleic acid or EtOH (control) for 24 hrs followed by fixation and immunolabeling of phospholipids and neutral lipids. Scale bar: 10 µm. Two confocal planes are shown for oleic acid treatment. D, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells treated with Torin1 and BafA1 or ATG7 inhibitor were fixed and immunolabeled with an anti-HA antibody. Scale bar: 10 µm. Arrowheads indicate HA-NeonGreen colocalization events.

    Journal: bioRxiv

    Article Title: ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

    doi: 10.1101/2020.01.01.892521

    Figure Lengend Snippet: Validation of GABARAPL2 endoHA /ACSL3 endoNeonGreen cells. A, GABARAPL2 endoHA and GABARAPL2 endoHA /ACSL3 endoNeonGreen cells as well as parental HeLa cells transiently transfected with TOMM20-NeonGreen were lysed and analyzed by immunoblotting with indicated antibodies. B, Fixed GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were immunostained with an anti-calnexin antibody. Scale bar: 10 µm. C, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells were treated with oleic acid or EtOH (control) for 24 hrs followed by fixation and immunolabeling of phospholipids and neutral lipids. Scale bar: 10 µm. Two confocal planes are shown for oleic acid treatment. D, GABARAPL2 endoHA /ACSL3 endoNeonGreen cells treated with Torin1 and BafA1 or ATG7 inhibitor were fixed and immunolabeled with an anti-HA antibody. Scale bar: 10 µm. Arrowheads indicate HA-NeonGreen colocalization events.

    Article Snippet: The following reagents were used for treatments: oleic acid (EMD Millipore, 4954, 600 µm in EtOH, 30 min or 24 hrs), Bafilomycin A1 (Biomol, Cay11038-1, 200 nM in DMSO, 2 hrs), Torin 1 (Tocris, 4247, 250 nM in DMSO, 2 hrs), Bortezomib (LC Labs B-1408, 1 µM in PBS, 8 hrs), ATG7 inhibitor (Takeda ML00792183, 1 µM, 24 hrs).

    Techniques: Transfection, Immunolabeling

    Influences of ACSL3 on the ufmylation pathway. A,B, GABARAPL2 endoHA cells were transfected with ACSL3 siRNAs and treated with Btz or BafA1 followed by lysis and immunoblot analysis using indicated antibodies. C, GABARAPL2 endoHA cells were treated with oleic acid for 30 min or 24 hrs or with EtOH for 24 hrs prior to lysis and immunoblotting with indicated antibodies. D, Working model summarizing the impact of ACSL3 and LD biogenesis on the ufmylation pathway. Upon recruitment via GABARAPL2, UBA5 is anchored at the ER membrane by ACSL3. LD induction through oleic acid blocks ufmylation through degradation-mediated disassembly of the UFM1 E3 enzyme complex. Completion of LD formation leads to reassemble of the E3 complex and increased ufmylation. Dotted blue arrows indicate ER-recruitment, black arrows indicate ufmylation cascade.

    Journal: bioRxiv

    Article Title: ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

    doi: 10.1101/2020.01.01.892521

    Figure Lengend Snippet: Influences of ACSL3 on the ufmylation pathway. A,B, GABARAPL2 endoHA cells were transfected with ACSL3 siRNAs and treated with Btz or BafA1 followed by lysis and immunoblot analysis using indicated antibodies. C, GABARAPL2 endoHA cells were treated with oleic acid for 30 min or 24 hrs or with EtOH for 24 hrs prior to lysis and immunoblotting with indicated antibodies. D, Working model summarizing the impact of ACSL3 and LD biogenesis on the ufmylation pathway. Upon recruitment via GABARAPL2, UBA5 is anchored at the ER membrane by ACSL3. LD induction through oleic acid blocks ufmylation through degradation-mediated disassembly of the UFM1 E3 enzyme complex. Completion of LD formation leads to reassemble of the E3 complex and increased ufmylation. Dotted blue arrows indicate ER-recruitment, black arrows indicate ufmylation cascade.

    Article Snippet: The following reagents were used for treatments: oleic acid (EMD Millipore, 4954, 600 µm in EtOH, 30 min or 24 hrs), Bafilomycin A1 (Biomol, Cay11038-1, 200 nM in DMSO, 2 hrs), Torin 1 (Tocris, 4247, 250 nM in DMSO, 2 hrs), Bortezomib (LC Labs B-1408, 1 µM in PBS, 8 hrs), ATG7 inhibitor (Takeda ML00792183, 1 µM, 24 hrs).

    Techniques: Transfection, Lysis

    Interaction analysis of endogenously tagged hATG8 proteins. A , GABARAPL2 endoHA and parental HeLa cell lysates were analyzed by immunoblotting using anti-HA and -PCNA antibodies. The latter was used as loading control. B,C , GABARAPL2 endoHA cells were reversely transfected for 72 hrs with non-targeting (sicrtl) or GABARAPL2 siRNA followed by lysis and immunoblot analysis ( B ) or fixation and immunolabeling ( C ) using an anti-HA antibody. Scale bar: 10 µm. D,E, GABARAPL2 endoHA cells were treated as indicated and subjected to lysis and immunoblotting ( D ) or fixation and immunolabeling ( E ) using anti-HA and -p62 antibodies. Scale bar: 10 µm. Arrowheads indicate colocalization events. F, Scatterplot represents interaction proteomics of SILAC labeled GABARAPL2 endoHA cells differentially treated with Torin1 and BafA1 (light) or ATG7 inhibitor (heavy). Significantly enriched proteins upon Torin1 and BafA1 combination treatment or ATG7 inhibition are highlighted in red and blue, respectively. Proteins in grey are unchanged. G-I, Immunoblot analysis of anti-HA immunoprecipitates from lysates derived from parental HeLa and GABARAPL2 endoHA cells transiently transfected for 48 hrs with myc-tagged ATG7 ( G ), p62 ( H ) or ACSL3 ( I ).

    Journal: bioRxiv

    Article Title: ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

    doi: 10.1101/2020.01.01.892521

    Figure Lengend Snippet: Interaction analysis of endogenously tagged hATG8 proteins. A , GABARAPL2 endoHA and parental HeLa cell lysates were analyzed by immunoblotting using anti-HA and -PCNA antibodies. The latter was used as loading control. B,C , GABARAPL2 endoHA cells were reversely transfected for 72 hrs with non-targeting (sicrtl) or GABARAPL2 siRNA followed by lysis and immunoblot analysis ( B ) or fixation and immunolabeling ( C ) using an anti-HA antibody. Scale bar: 10 µm. D,E, GABARAPL2 endoHA cells were treated as indicated and subjected to lysis and immunoblotting ( D ) or fixation and immunolabeling ( E ) using anti-HA and -p62 antibodies. Scale bar: 10 µm. Arrowheads indicate colocalization events. F, Scatterplot represents interaction proteomics of SILAC labeled GABARAPL2 endoHA cells differentially treated with Torin1 and BafA1 (light) or ATG7 inhibitor (heavy). Significantly enriched proteins upon Torin1 and BafA1 combination treatment or ATG7 inhibition are highlighted in red and blue, respectively. Proteins in grey are unchanged. G-I, Immunoblot analysis of anti-HA immunoprecipitates from lysates derived from parental HeLa and GABARAPL2 endoHA cells transiently transfected for 48 hrs with myc-tagged ATG7 ( G ), p62 ( H ) or ACSL3 ( I ).

    Article Snippet: The following reagents were used for treatments: oleic acid (EMD Millipore, 4954, 600 µm in EtOH, 30 min or 24 hrs), Bafilomycin A1 (Biomol, Cay11038-1, 200 nM in DMSO, 2 hrs), Torin 1 (Tocris, 4247, 250 nM in DMSO, 2 hrs), Bortezomib (LC Labs B-1408, 1 µM in PBS, 8 hrs), ATG7 inhibitor (Takeda ML00792183, 1 µM, 24 hrs).

    Techniques: Transfection, Lysis, Immunolabeling, Labeling, Inhibition, Derivative Assay

    Sym004 promotes ubiquitin-independent, ESCRT-dependent lysosomal degradation of EGFR. (a) SCC47 cells were serum-starved for 1 h, then MG132 (10 µM) or bafilomycin A1 (600 nM) were added, followed 1 h later by cisplatin (50 µM), cetuximab, Sym004 or EGF. The cells were incubated overnight and lysed with RIPA buffer. (b) SCC47 cells were serum-starved for 1 h, then bafilomycin A1 (600 nM) was added followed 1 h later by Sym004-AF568 or EGF-AF647. The cells were incubated overnight, fixed and stained with EGFR-AF488 antibody. (c) Serum-starved SCC47 were treated with cetuximab, Sym004 or EGF as indicated, then lysed. EGFR was immunoprecipitated with anti-EGFR antibody (normal mouse IgG used in Control). For quantifications, ubiquitylated EGFR was normalised to total EGFR. (d) Serum-starved, Hrs- or Tsg101-depleted (or control) SCC47 cells were treated with Sym004 (3 µg/ml or 30 µg/ml) or EGF for 6 h, then lysed. (e) The cells treated as in (d) were fixed and stained with EGFR-AF488. Nuclei were stained with Hoechst 33342. All immunoblots were cropped for clarity.

    Journal: Scientific Reports

    Article Title: Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

    doi: 10.1038/s41598-019-57153-9

    Figure Lengend Snippet: Sym004 promotes ubiquitin-independent, ESCRT-dependent lysosomal degradation of EGFR. (a) SCC47 cells were serum-starved for 1 h, then MG132 (10 µM) or bafilomycin A1 (600 nM) were added, followed 1 h later by cisplatin (50 µM), cetuximab, Sym004 or EGF. The cells were incubated overnight and lysed with RIPA buffer. (b) SCC47 cells were serum-starved for 1 h, then bafilomycin A1 (600 nM) was added followed 1 h later by Sym004-AF568 or EGF-AF647. The cells were incubated overnight, fixed and stained with EGFR-AF488 antibody. (c) Serum-starved SCC47 were treated with cetuximab, Sym004 or EGF as indicated, then lysed. EGFR was immunoprecipitated with anti-EGFR antibody (normal mouse IgG used in Control). For quantifications, ubiquitylated EGFR was normalised to total EGFR. (d) Serum-starved, Hrs- or Tsg101-depleted (or control) SCC47 cells were treated with Sym004 (3 µg/ml or 30 µg/ml) or EGF for 6 h, then lysed. (e) The cells treated as in (d) were fixed and stained with EGFR-AF488. Nuclei were stained with Hoechst 33342. All immunoblots were cropped for clarity.

    Article Snippet: Other reagents included: Human EGF (E9644, Sigma-Aldrich, used at 50 ng/ml, unless otherwise stated), EGF Biotinylated, AF647-conjugated (E35351, Thermo Fisher Scientific), nProtein A Sepharose 4 Fast Flow (GE17–5280–01, GE Healthcare), Fluorescein-conjugated Dextran 70 kDa (D1822, Thermo Fisher Scientific), Hoechst 33342 (H3570, Thermo Fisher Scientific), plasmids wt dynamin 1 pEGFP (Addgene #34680) and K44A Dynamin 1 pEGFP (Addgene #34681) were generated by Sandra Schmid (UT Southwestern Medical Center, TX, USA), Lipofectamine 3000 (L3000008, Thermo Fisher Scientific), Lipofectamine RNAiMAX (13778075, Thermo Fisher Scientific), p38 inhibitor SB203580 (559389, Merck, 10 µM, 30 min pre-treatment), Erlotinib (CDS022564, Sigma-Aldrich, 0.5 µM, 30 min pre-treatment, unless otherwise stated), Bafilomycin A1 (196000, Merck, 600 nM, 1 h or overnight, as indicated), MG132 (474791, Merck, 10 µM), Cytochalasin D (504776, Merck, 5 µM or 50 µM, 30 min pre-treatment unless otherwise stated), RIPA buffer (9806 S, Cell Signalling Technology, used throughout, unless otherwise stated), CellLytic M (C2978, Sigma-Aldrich), Laemmli (0.0625 M Tris/HCl pH 6.8, 2% SDS, 10% glycerol).

    Techniques: Incubation, Staining, Immunoprecipitation, Western Blot

    Sym004 forms distinctive cell surface structures and promotes EGFR localisation within a detergent-insoluble fraction. (a) SCC47 cells were serum-starved for 2 h, then incubated with cetuximab or Sym004 overnight, fixed and immunostained with α-EGFR antibody. Nuclei stained with Hoechst 33342. (b) SCC47 cells were serum-starved for 2 h and treated with cetuximab or Sym004 for 5 h, then lysed with CellLytic M. (c) SCC47 cells were serum-starved, final 2 h in the presence of LysoTracker Red (100 nM), then imaged live in cell-imaging medium (CIM) upon addition of Sym004-AF488. The images were acquired every 3 min and generated from 9 z-stacks with 0.424 µm intervals. Yellow arrowheads indicate colocalisation between Sym004 and LysoTracker and white arrowheads point to the elongated tubular structures. (d) GFP-CLC cells were serum-starved for a minimum 2 h and imaged live in the presence of EGF-Cy3b. For Sym004, serum-starved cells were pre-treated for 5 min with Sym004-AF568 (10 µg/ml), washed with PBS and imaged for indicated times. (e) Cells were treated with Sym004-10 nm Gold for 15 min before processing for electron microscopy. Gold was found in a variety of tubules connected with the cell surface (arrowheads), as well as pits and vesicles (arrows) both coated (white) and uncoated (black). (f) SCC47 cells were serum-starved, final 1 h in the presence of MG132 (10 µM), bafilomycin A1 (600 nM) or DMSO (control), then treated with Sym004 or EGF for 2 h. The cells were lysed with CellLytic M. For quantifications, EGFR was normalised to calnexin. (g) Serum-starved SCC47 cells were treated with Sym004 or EGF for 2 h, then lysed with different buffers. (h) Serum-starved SCC47 cells were incubated on ice with cetuximab, Sym004 or EGF for 30 min. The cells were then either lysed (CellLytic M) or placed for further 2 h at 37 °C, then lysed. Error bars, SEM, **p

    Journal: Scientific Reports

    Article Title: Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

    doi: 10.1038/s41598-019-57153-9

    Figure Lengend Snippet: Sym004 forms distinctive cell surface structures and promotes EGFR localisation within a detergent-insoluble fraction. (a) SCC47 cells were serum-starved for 2 h, then incubated with cetuximab or Sym004 overnight, fixed and immunostained with α-EGFR antibody. Nuclei stained with Hoechst 33342. (b) SCC47 cells were serum-starved for 2 h and treated with cetuximab or Sym004 for 5 h, then lysed with CellLytic M. (c) SCC47 cells were serum-starved, final 2 h in the presence of LysoTracker Red (100 nM), then imaged live in cell-imaging medium (CIM) upon addition of Sym004-AF488. The images were acquired every 3 min and generated from 9 z-stacks with 0.424 µm intervals. Yellow arrowheads indicate colocalisation between Sym004 and LysoTracker and white arrowheads point to the elongated tubular structures. (d) GFP-CLC cells were serum-starved for a minimum 2 h and imaged live in the presence of EGF-Cy3b. For Sym004, serum-starved cells were pre-treated for 5 min with Sym004-AF568 (10 µg/ml), washed with PBS and imaged for indicated times. (e) Cells were treated with Sym004-10 nm Gold for 15 min before processing for electron microscopy. Gold was found in a variety of tubules connected with the cell surface (arrowheads), as well as pits and vesicles (arrows) both coated (white) and uncoated (black). (f) SCC47 cells were serum-starved, final 1 h in the presence of MG132 (10 µM), bafilomycin A1 (600 nM) or DMSO (control), then treated with Sym004 or EGF for 2 h. The cells were lysed with CellLytic M. For quantifications, EGFR was normalised to calnexin. (g) Serum-starved SCC47 cells were treated with Sym004 or EGF for 2 h, then lysed with different buffers. (h) Serum-starved SCC47 cells were incubated on ice with cetuximab, Sym004 or EGF for 30 min. The cells were then either lysed (CellLytic M) or placed for further 2 h at 37 °C, then lysed. Error bars, SEM, **p

    Article Snippet: Other reagents included: Human EGF (E9644, Sigma-Aldrich, used at 50 ng/ml, unless otherwise stated), EGF Biotinylated, AF647-conjugated (E35351, Thermo Fisher Scientific), nProtein A Sepharose 4 Fast Flow (GE17–5280–01, GE Healthcare), Fluorescein-conjugated Dextran 70 kDa (D1822, Thermo Fisher Scientific), Hoechst 33342 (H3570, Thermo Fisher Scientific), plasmids wt dynamin 1 pEGFP (Addgene #34680) and K44A Dynamin 1 pEGFP (Addgene #34681) were generated by Sandra Schmid (UT Southwestern Medical Center, TX, USA), Lipofectamine 3000 (L3000008, Thermo Fisher Scientific), Lipofectamine RNAiMAX (13778075, Thermo Fisher Scientific), p38 inhibitor SB203580 (559389, Merck, 10 µM, 30 min pre-treatment), Erlotinib (CDS022564, Sigma-Aldrich, 0.5 µM, 30 min pre-treatment, unless otherwise stated), Bafilomycin A1 (196000, Merck, 600 nM, 1 h or overnight, as indicated), MG132 (474791, Merck, 10 µM), Cytochalasin D (504776, Merck, 5 µM or 50 µM, 30 min pre-treatment unless otherwise stated), RIPA buffer (9806 S, Cell Signalling Technology, used throughout, unless otherwise stated), CellLytic M (C2978, Sigma-Aldrich), Laemmli (0.0625 M Tris/HCl pH 6.8, 2% SDS, 10% glycerol).

    Techniques: Incubation, Staining, Imaging, Generated, Electron Microscopy

    Modulation of nonclassical secretory pathway by BafA1 treatment in H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T. ( A ) Scheme of markers for distinct nonclassical secretory pathways including exosome release, endocytic recycling pathway, and microparticle shedding. MVBs = multivesicular bodies, ILVs = intraluminal vesicles, PS = phosphatidylserine ( B ) Quantification of extracellular CD63 levels in the medium of H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP compared to control cells (mock) by dot blot analysis. Cells have been treated with or without 200 nM BafA1 for 12 h. ( C ) Quantification of RAB11A expression in H4 cell lysates expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP -/+ BafA1 treatment. ( D ) Left: FACS analysis of released microparticles by transfected H4 cells -/+ BafA1 using a FITC-labeled ANXA5 antibody. Right: Representative scatter plots and histograms showing medium with ANXA5 as control for background signals, unstained microparticles to exclude auto fluorescence and microparticles stained for ANXA5 as positive control defining gating criteria. All values are mean + s.e.m; differences are significant at (*) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: Modulation of nonclassical secretory pathway by BafA1 treatment in H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T. ( A ) Scheme of markers for distinct nonclassical secretory pathways including exosome release, endocytic recycling pathway, and microparticle shedding. MVBs = multivesicular bodies, ILVs = intraluminal vesicles, PS = phosphatidylserine ( B ) Quantification of extracellular CD63 levels in the medium of H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP compared to control cells (mock) by dot blot analysis. Cells have been treated with or without 200 nM BafA1 for 12 h. ( C ) Quantification of RAB11A expression in H4 cell lysates expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP -/+ BafA1 treatment. ( D ) Left: FACS analysis of released microparticles by transfected H4 cells -/+ BafA1 using a FITC-labeled ANXA5 antibody. Right: Representative scatter plots and histograms showing medium with ANXA5 as control for background signals, unstained microparticles to exclude auto fluorescence and microparticles stained for ANXA5 as positive control defining gating criteria. All values are mean + s.e.m; differences are significant at (*) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Expressing, Dot Blot, FACS, Transfection, Labeling, Fluorescence, Staining, Positive Control

    Toxic response of naïve H4 cells exposed to extracellular SNCA. ( A ) Immunocytochemistry of CASP3 + cells after 6 h exposure of naïve H4 cells to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, and both SNCA-T and SNCAIP (and control H4 cells). Scale bar 40 μm. ( B ) Increase of toxicity in naïve H4 cells exposed to PFs containing extracellular SNCA from untreated and BafA1-treated cells after 6 h compared to exposure with control medium measured by AK release using ToxiLight assay. ( C ) Higher magnification of CASP3 + cells reveals an association to the surface of exposed H4 cells and intracellular accumulation of SNCA + PFs. Scale bar 5 μm. All values are mean + s.e.m; differences were significant at (*) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: Toxic response of naïve H4 cells exposed to extracellular SNCA. ( A ) Immunocytochemistry of CASP3 + cells after 6 h exposure of naïve H4 cells to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, and both SNCA-T and SNCAIP (and control H4 cells). Scale bar 40 μm. ( B ) Increase of toxicity in naïve H4 cells exposed to PFs containing extracellular SNCA from untreated and BafA1-treated cells after 6 h compared to exposure with control medium measured by AK release using ToxiLight assay. ( C ) Higher magnification of CASP3 + cells reveals an association to the surface of exposed H4 cells and intracellular accumulation of SNCA + PFs. Scale bar 5 μm. All values are mean + s.e.m; differences were significant at (*) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Immunocytochemistry, Transfection

    Neuronal loss paralleled by astro- and microgliosis in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of neuronal loss and astrogliosis paralleled by a microgliosis. ( A ) Representative images of RBFOX3/NeuN + neurons, GFAP + astroglia and the microglial marker AIF1 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. The insets in the 1st row depict the region of quantification. Insets in the 2nd row depict regions which are magnified in the 3rd row. Scale bar for RBFOX3/NeuN in the hippocampus 50 μm, GFAP in the hippocampus 50 μm and 20 μm (2nd and 3rd row) and AIF1 in the hippocampus and the neocortex 20 μm (4th and 5th row). Quantification of cell number and optical density quantification of ( B ) the neuronal marker RBFOX3/NeuN + , ( C ) the astroglial marker GFAP, ( D ) the microglial marker AIF1 in the hippocampus, and (E) AIF1 in the neocortex of SNCA-tg mice compared to non-tg mice. ( B ) to ( E ) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. All values are mean + s.e.m; differences are significant at (#) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: Neuronal loss paralleled by astro- and microgliosis in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of neuronal loss and astrogliosis paralleled by a microgliosis. ( A ) Representative images of RBFOX3/NeuN + neurons, GFAP + astroglia and the microglial marker AIF1 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. The insets in the 1st row depict the region of quantification. Insets in the 2nd row depict regions which are magnified in the 3rd row. Scale bar for RBFOX3/NeuN in the hippocampus 50 μm, GFAP in the hippocampus 50 μm and 20 μm (2nd and 3rd row) and AIF1 in the hippocampus and the neocortex 20 μm (4th and 5th row). Quantification of cell number and optical density quantification of ( B ) the neuronal marker RBFOX3/NeuN + , ( C ) the astroglial marker GFAP, ( D ) the microglial marker AIF1 in the hippocampus, and (E) AIF1 in the neocortex of SNCA-tg mice compared to non-tg mice. ( B ) to ( E ) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. All values are mean + s.e.m; differences are significant at (#) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Transgenic Assay, Mouse Assay, Immunohistochemistry, Marker

    ( See previous page ). Characterization of particle fractions (PFs) prepared from conditioned medium of transfected H4 cells. ( A ) to ( F ) Western blot and dot blot analysis of SNCA associated with PFs prepared from conditioned medium of H4 cells expressing high-aggregating SNCA-T and SNCAIP or low-aggregating SNCA. ( A ) Representative protein gel blots of total SNCA + PFs prepared from 12 ml conditioned medium of transfected H4 cells -/+BafA1 show an increased presence of extracellular SNCA (14 kDa) or SNCA-T (27 kDa) after BafA1 treatment. ( B ) 12 ml supernatant after PF separation contain only low levels of soluble SNCA. ( C ) Representative western blots of SNCA PFs -/+BafA1 after filter retardation to exclude particles of different size by using distinct pore size filters reveal enlarged particles ( > 1 .2 μm) in the presence of BafA1. ( D ) Representative dot blots of SNCA in the conditioned medium of H4 cells expressing low-aggregating SNCA -/+BafA1. ( E ) Representative protein gel blots of SNCA-T and SNCAIP PFs after exclusion of particles of different size by using distinct pore size filters. ( F ) Quantification of SNCA associated with PFs after using filters of defined pore size normalized to SNCA levels in unfiltered PFs displaying a shift toward smaller particles by BafA1. ( G ) BafA1 effect on oligomerization analysis of SNCA associated with PFs prepared from H4 cells expressing SNCA, as well as SNCA-T and SNCAIP measured by sucrose gradient centrifugation. All values are mean + s.e.m. Differences are significant at (*) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: ( See previous page ). Characterization of particle fractions (PFs) prepared from conditioned medium of transfected H4 cells. ( A ) to ( F ) Western blot and dot blot analysis of SNCA associated with PFs prepared from conditioned medium of H4 cells expressing high-aggregating SNCA-T and SNCAIP or low-aggregating SNCA. ( A ) Representative protein gel blots of total SNCA + PFs prepared from 12 ml conditioned medium of transfected H4 cells -/+BafA1 show an increased presence of extracellular SNCA (14 kDa) or SNCA-T (27 kDa) after BafA1 treatment. ( B ) 12 ml supernatant after PF separation contain only low levels of soluble SNCA. ( C ) Representative western blots of SNCA PFs -/+BafA1 after filter retardation to exclude particles of different size by using distinct pore size filters reveal enlarged particles ( > 1 .2 μm) in the presence of BafA1. ( D ) Representative dot blots of SNCA in the conditioned medium of H4 cells expressing low-aggregating SNCA -/+BafA1. ( E ) Representative protein gel blots of SNCA-T and SNCAIP PFs after exclusion of particles of different size by using distinct pore size filters. ( F ) Quantification of SNCA associated with PFs after using filters of defined pore size normalized to SNCA levels in unfiltered PFs displaying a shift toward smaller particles by BafA1. ( G ) BafA1 effect on oligomerization analysis of SNCA associated with PFs prepared from H4 cells expressing SNCA, as well as SNCA-T and SNCAIP measured by sucrose gradient centrifugation. All values are mean + s.e.m. Differences are significant at (*) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Western Blot, Dot Blot, Expressing, Gradient Centrifugation

    BafA1 treatment modulates aggregate formation and levels of extracellular SNCA in transfected H4 cells. ( A ) to ( C ): Immunocytochemistry of H4 cells transfected with SNCA, SNCA-T and SNCA-T and SNCAIP: ( A ) Large aggregates of different size and morphology can be observed after transfection with high-aggregating SNCA-T and SNCAIP whereas SNCA only occasionally leads to smaller intracellular aggregates sized below 1 μm. Scale bar: 5 μm. ( B ) Quantification of differentially sized aggregates per cell in H4 cells transfected with SNCA-T alone compared to cotransfection of SNCA-T with SNCAIP. ( C ) Treatment with 200 nM BafA1 for 12 h results in smaller and more punctate intracellular structures. ( D ) to ( F ): Dot blot analysis of extracellular SNCA in the conditioned medium of H4 neuron-like cells and CG4 oligodendroglial cells: ( D ) Representative dot blots of H4 cell medium containing extracellular SNCA. Recombinant SNCA is used as quantification standard. ( E ) Dot blot quantification of extracellular SNCA levels in similar volumes of conditioned medium of H4 cells expressing high-aggregating SNCA-T compared to low-aggregating SNCA and mock-transfected control cells 36 h post-transfection. Treatment with 200 nM BafA1 for 12 h results in slightly increased levels of extracellular SNCA in the medium of H4 cells transfected with SNCA, and both SNCA-T and SNCAIP compared to untreated cells. ( F ) Representative dot blots showing a lack of extracellular SNCA in supernatants of undifferentiated and 6 d differentiated rat oligodendroglial CG4 cells overexpressing SNCA to exclude nonspecific SNCA release. All values are presented as mean + s.e.m; (*) P = 0.009.

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: BafA1 treatment modulates aggregate formation and levels of extracellular SNCA in transfected H4 cells. ( A ) to ( C ): Immunocytochemistry of H4 cells transfected with SNCA, SNCA-T and SNCA-T and SNCAIP: ( A ) Large aggregates of different size and morphology can be observed after transfection with high-aggregating SNCA-T and SNCAIP whereas SNCA only occasionally leads to smaller intracellular aggregates sized below 1 μm. Scale bar: 5 μm. ( B ) Quantification of differentially sized aggregates per cell in H4 cells transfected with SNCA-T alone compared to cotransfection of SNCA-T with SNCAIP. ( C ) Treatment with 200 nM BafA1 for 12 h results in smaller and more punctate intracellular structures. ( D ) to ( F ): Dot blot analysis of extracellular SNCA in the conditioned medium of H4 neuron-like cells and CG4 oligodendroglial cells: ( D ) Representative dot blots of H4 cell medium containing extracellular SNCA. Recombinant SNCA is used as quantification standard. ( E ) Dot blot quantification of extracellular SNCA levels in similar volumes of conditioned medium of H4 cells expressing high-aggregating SNCA-T compared to low-aggregating SNCA and mock-transfected control cells 36 h post-transfection. Treatment with 200 nM BafA1 for 12 h results in slightly increased levels of extracellular SNCA in the medium of H4 cells transfected with SNCA, and both SNCA-T and SNCAIP compared to untreated cells. ( F ) Representative dot blots showing a lack of extracellular SNCA in supernatants of undifferentiated and 6 d differentiated rat oligodendroglial CG4 cells overexpressing SNCA to exclude nonspecific SNCA release. All values are presented as mean + s.e.m; (*) P = 0.009.

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Transfection, Immunocytochemistry, Cotransfection, Dot Blot, Recombinant, Expressing

    Toxic response of primary neuronal cultures exposed to extracellular SNCA. ( A ) Exposure of primary hippocampal neuronal cultures (TUBB3, neuronal marker; GFAP, astroglial marker) to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, SNCA-T and SNCAIP (and control H4 cells). ( B ) The toxic response of primary neurons exposed to PFs from untreated and BafA1-treated H4 cells after 6 h exposure time measured by AK release using ToxiLight assay. ( C ) Exposure of primary neuronal cultures to SNCA, as well as SNCA-T and SNCAIP PFs leads to the formation of intracellular SNCA + accumulations in TUBB3 + neuronal cells. Z-stack of confocal images, scale bar 10 μm. All values are mean + s.e.m. Differences were significant at (*) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: Toxic response of primary neuronal cultures exposed to extracellular SNCA. ( A ) Exposure of primary hippocampal neuronal cultures (TUBB3, neuronal marker; GFAP, astroglial marker) to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, SNCA-T and SNCAIP (and control H4 cells). ( B ) The toxic response of primary neurons exposed to PFs from untreated and BafA1-treated H4 cells after 6 h exposure time measured by AK release using ToxiLight assay. ( C ) Exposure of primary neuronal cultures to SNCA, as well as SNCA-T and SNCAIP PFs leads to the formation of intracellular SNCA + accumulations in TUBB3 + neuronal cells. Z-stack of confocal images, scale bar 10 μm. All values are mean + s.e.m. Differences were significant at (*) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Marker, Transfection

    Inflammatory response in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of the inflammatory response in SNCA transgenic mice. ( A ) Representative images of the inflammatory marker TNF and IL6 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. Black arrows indicate TNF and IL6 immunoreactivity. Scale bar for TNF and IL6 in the hippocampus 30 μm. Optical density quantification of ( B ) the inflammatory markers TNF and ( D ) IL6 in the hippocampus of SNCA-tg mice compared to non-tg mice. ( B ) to ( D ) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. ( C ) The absolute amount of TNF as measured by ELISA in SNCA-tg mice treated with BafA1 compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: Inflammatory response in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of the inflammatory response in SNCA transgenic mice. ( A ) Representative images of the inflammatory marker TNF and IL6 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. Black arrows indicate TNF and IL6 immunoreactivity. Scale bar for TNF and IL6 in the hippocampus 30 μm. Optical density quantification of ( B ) the inflammatory markers TNF and ( D ) IL6 in the hippocampus of SNCA-tg mice compared to non-tg mice. ( B ) to ( D ) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. ( C ) The absolute amount of TNF as measured by ELISA in SNCA-tg mice treated with BafA1 compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Transgenic Assay, Mouse Assay, Immunohistochemistry, Marker, Enzyme-linked Immunosorbent Assay

    ( See previous page ). Modulation of intracellular and extracellular SNCA in transgenic mice, by BafA1. ( A ) Immunohistochemistry of transgenic mice overexpressing human SNCA under the control of the PDGFB -promoter (SNCA-tg) and nontransgenic mice (non-tg) using an antibody recognizing human and mouse SNCA. Transgenic mice display SNCA + inclusions in neuronal bodies and in the neuropil of the neocortex (Neoctx) compared to non-tg mice. BafA1 has been applied systemically by daily intraperitoneal injection of 0.3 mg/kg or vehicle (saline) for 5 consecutive days. The inset indicates the representative region of neocortex depicted in the 2nd and in the 3rd row. Scale bar 200 μm. Transgenic mice display SNCA immunoreactivity in the molecular layer (ML) of the hippocampus (Hippo) indicated by black arrows. Scale bar 50 μm. CA1: cornu ammonis field1; SL: stratum lacunosum ( B ) BafA1 treatment of SNCA-tg mice diminishes the number of neurons bearing SNCA + inclusions within the neocortex compared to vehicle-treated SNCA-tg mice. ( C ) Quantification of the percentage of the neocortical neuropil area that shows SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. ( D ) Percentage of the neuropil area in the hippocampal ML that displays SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. ( E ) Left: Representative western blots of protein lysates probed for endogenous mouse and human SNCA depict monomeric (14 kDa) and oligomeric species (42 and 56 kDa). ACTB serves as loading control (42 kDa). Right: Western blot quantification of SNCA expression levels in BafA1-treated transgenic mice overexpressing human SNCA and normalized to ACTB (n = 3 animals per group). ( F ) Extracellular SNCA levels in the CSF of transgenic mice overexpressing SNCA measured by ELISA. All values are mean + s.e.m. Differences are significant at (#) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: ( See previous page ). Modulation of intracellular and extracellular SNCA in transgenic mice, by BafA1. ( A ) Immunohistochemistry of transgenic mice overexpressing human SNCA under the control of the PDGFB -promoter (SNCA-tg) and nontransgenic mice (non-tg) using an antibody recognizing human and mouse SNCA. Transgenic mice display SNCA + inclusions in neuronal bodies and in the neuropil of the neocortex (Neoctx) compared to non-tg mice. BafA1 has been applied systemically by daily intraperitoneal injection of 0.3 mg/kg or vehicle (saline) for 5 consecutive days. The inset indicates the representative region of neocortex depicted in the 2nd and in the 3rd row. Scale bar 200 μm. Transgenic mice display SNCA immunoreactivity in the molecular layer (ML) of the hippocampus (Hippo) indicated by black arrows. Scale bar 50 μm. CA1: cornu ammonis field1; SL: stratum lacunosum ( B ) BafA1 treatment of SNCA-tg mice diminishes the number of neurons bearing SNCA + inclusions within the neocortex compared to vehicle-treated SNCA-tg mice. ( C ) Quantification of the percentage of the neocortical neuropil area that shows SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. ( D ) Percentage of the neuropil area in the hippocampal ML that displays SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. ( E ) Left: Representative western blots of protein lysates probed for endogenous mouse and human SNCA depict monomeric (14 kDa) and oligomeric species (42 and 56 kDa). ACTB serves as loading control (42 kDa). Right: Western blot quantification of SNCA expression levels in BafA1-treated transgenic mice overexpressing human SNCA and normalized to ACTB (n = 3 animals per group). ( F ) Extracellular SNCA levels in the CSF of transgenic mice overexpressing SNCA measured by ELISA. All values are mean + s.e.m. Differences are significant at (#) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Polyacrylamide Gel Electrophoresis, Transgenic Assay, Mouse Assay, Immunohistochemistry, Injection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    BafA1 treatment increases the accumulation of SNCA in glial cells in the microenvironment of SNCA-expressing neurons in transgenic mice. Immunohistochemical analysis of glial cells (S100B + ) in the microenvironment of SNCA + neurons within the colliculus and the hippocampus of SNCA-tg mice. ( A ) SNCA immunoreactivity in glial cells in the colliculus of SNCA-tg mice compared to non-tg mice -/+BafA1 treatment. Insets in the 1st row indicate the region within the superior colliculus which is magnified in the 2nd row. Black arrowheads indicate the presence of SNCA + neurons based on their morphological appearance in close proximity. Scale bar 1st row 50 μm, 2nd and 3rd row 20 μm. ( B ) Quantification of glial cells based on their morphology displaying SNCA immunoreactivity in the colliculus of SNCA-tg mice -/+BafA1 treatment. ( C ) Confocal images of S100B + astroglia (red) double-labeling for SNCA (green) in close proximity to SNCA + neurons (N; defined by morphology) in the hippocampus and colliculus of SNCA-tg mice either treated with BafA1 or vehicle. ( D ) and ( E ) Quantification of S100B + and SNCA + cells after BafA1 treatment in SNCA-tg mice compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P

    Journal: Autophagy

    Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

    doi: 10.4161/auto.36436

    Figure Lengend Snippet: BafA1 treatment increases the accumulation of SNCA in glial cells in the microenvironment of SNCA-expressing neurons in transgenic mice. Immunohistochemical analysis of glial cells (S100B + ) in the microenvironment of SNCA + neurons within the colliculus and the hippocampus of SNCA-tg mice. ( A ) SNCA immunoreactivity in glial cells in the colliculus of SNCA-tg mice compared to non-tg mice -/+BafA1 treatment. Insets in the 1st row indicate the region within the superior colliculus which is magnified in the 2nd row. Black arrowheads indicate the presence of SNCA + neurons based on their morphological appearance in close proximity. Scale bar 1st row 50 μm, 2nd and 3rd row 20 μm. ( B ) Quantification of glial cells based on their morphology displaying SNCA immunoreactivity in the colliculus of SNCA-tg mice -/+BafA1 treatment. ( C ) Confocal images of S100B + astroglia (red) double-labeling for SNCA (green) in close proximity to SNCA + neurons (N; defined by morphology) in the hippocampus and colliculus of SNCA-tg mice either treated with BafA1 or vehicle. ( D ) and ( E ) Quantification of S100B + and SNCA + cells after BafA1 treatment in SNCA-tg mice compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P

    Article Snippet: The transgenic animals and nontransgenic littermates at the age of 5 to 6 mo were treated with BafA1 (0.3 mg/kg) or vehicle (saline) by daily intraperitoneal injections for 5 consecutive d (4 groups, 8 animals each).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Immunohistochemistry, Labeling