Journal: International Journal of Molecular Sciences

Article Title: Zinc Attenuates the Cytotoxicity of Some Stimuli by Reducing Endoplasmic Reticulum Stress in Hepatocytes

doi: 10.3390/ijms20092192

Figure Lengend Snippet: Effect of copper on autophagy. ( A ) OUMS-29 cells were treated with copper at concentrations of 100 or 300 μM for 12 h. Densitometry analysis of indicated proteins is shown on the right. ( B ) Light chain 3 (LC3) turnover assay. OUMS-29 cells were treated with copper at concentrations of 100 or 300 μM for 12 h with or without 50 nM of bafilomycin A1. Densitometry analysis of indicated proteins is shown on the right. ( C ) OUMS-29 cells were transfected with monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP) tandem fluorescent-tagged LC3 plasmids, and were treated with copper at concentrations of 100 or 300 μM for 12 h. Colocalizations of mRFP and GFP signals were measured by counting an overall total of 30 to 40 cells over the course of three independent experiments. Colocalization was shown as the percentage of merged signals in the total number of mRFP signals. Data are expressed as means ± SEM. ( D ) OUMS-29 cells were transfected with mRFP-LC3, and were treated with copper at concentrations of 100 or 300 μM for 12 h. Then, immunofluorescence staining for lysosomal-associated membrane protein 1 (Lamp1) was performed. ( E ) Effect of copper on lysosomal function. OUMS-29 cells, treated with copper at concentrations of 100 or 300 μM with or without bafilomycin A1 (50 nM) for 12 h, were loaded with 1 μM lysotracker Red for 1 h, followed by fixation, and then immunofluorescence staining for Lamp1 was performed. ( F ) Effect of copper on lysosomal function. OUMS-29 cells, treated with copper at concentrations of 100 or 300 μM with or without bafilomycin A1 (50 nM) for 12 h, were loaded with Magic Red Cathepsin B reagent for 1 h, and then analyzed by microscopy. ( G ) Huh7 cells were treated with copper at the concentration of 300 μM for 12 h. Densitometry analysis of indicated proteins is shown on the right. ( H ) Transmission electron micrograph of a Wilson disease patient. Arrowheads indicate the autophagosomes.

Article Snippet: The combinational evaluation of the same sample with or without treatment of bafilomycin A1 (Sigma-Aldrich) allows one to determine whether there has been an induction or inhibition of the autophagic flux.

Techniques: LC3 Turnover Assay, Transfection, Immunofluorescence, Staining, Microscopy, Concentration Assay, Transmission Assay

Journal: Scientific Reports

Article Title: Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy

doi: 10.1038/s41598-022-26775-x

Figure Lengend Snippet: Mitochondria are not efficiently degraded during PHB1 deficiency. Mode-K intestinal epithelial cells were transfected with two independent siPhb1 constructs (designated as a and b) or siNegative Control construct (siNC). ( A ) Cells were treated with 10 nM Bafilomycin A1 (BafA) for 1 h and markers of mitophagy (CoxIV, Tim50) were measured by western blot. ( B ) Mitophagic flux. ( C ) Relative mitochondrial/nuclear DNA level in Mode-K cells. ( D ) Cartoon depiction of mito-RFP-EGFP indicator of mitophagy. ( E , F ) Mode-K cells were co-transfected with mito-RFP-EGFP during siPhb1 knockdown and treated with 500 nM rotenone for 2 h to induce mitophagy. ( E ) IF staining of mito-RFP-EGFP. Scale bars: 10 μm, boxed pullout: 5 μm. ( F ) Quantitation of yellow and red pixel intensity using the average of 50 cells per well across 10 wells per treatment. Results are presented as individual data points ± SEM of 1 per treatment group performed in 3 independent experiments ( A , B ) or 5 per treatment group ( C ). * P < 0.05, ** P < 0.01, **** P < 0.001 vs. siNC veh by one-way ANOVA and Tukey’s posthoc test. See also Figs. and for full-length blots.

Article Snippet: Mitophagic flux was determined during Bafilomycin A1 treatment as described by Viva Detect Autophlux kit (VB3000, Viva Bioscience).

Techniques: Transfection, Construct, Western Blot, Staining, Quantitation Assay

Journal: EBioMedicine

Article Title: Tumor cells induce LAMP2a expression in tumor-associated macrophage for cancer progression

doi: 10.1016/j.ebiom.2019.01.045

Figure Lengend Snippet: LAMP2a targets PRDX1 and CRTC1 to modulate macrophages activation. (a) LAMP2a expression in TS-stimulated BMDMs which were treated by bafilomycin (Bafilo) or not. (b) Protein level of GPADH binding to LAMP2a in TS-stimulated BMDMs treated by bafilomycin (Bafilo) or not. (c) LAMP2a, PRDX1, CRTC1 and IRG1 expression in co-IP experiments of TS-stimulated BMDMs treated by bafilomycin (Bafilo) or not. All the IP-proteins were immunoprecipitated by anti-LAMP2a antibody and detected by respective antibodies. (d) Protein level of PRDX1, CRTC1 and IRG1 in TS-stimulated BMDMs which were transfected by sh-NC, sh-L2a or not. (e) The surface plasmon resonances (SPR) of LAMP2a-bound PRDX1, CRTC1 and IRG proteins. The results were fitted as dissociation constants (KDs). (f) Protein level of LAMP2a, PRDX1 and CRTC1 in TS-stimulated, genetically modified mouse HSCs-derived macrophages. “SCR” represents sg-SRC vector and “V” represents overexpression control vector. (g) Heatmap of relative mRNA expression of macrophage activation-related genes in mouse HSCs-derived macrophages described as ( f ). “pMIG” stands for overexpression control vector. Data were measured by qPCR, normalized to corresponding groups as noted, represented as log2 scale, with β-actin as control. (h) LDH release of 4 T1, CT26, LL/2 cells after 24 h co-culture with genetically modified HSCs-derived macrophages at a ratio of 40:1 as E:T. The percentage of cytotoxicity was calculated by maximum tumor cells LDH release control. (i) CREB and CEBP/β expression in TS-stimulated BMDMs treated by sh-NC, sh-L2a or not. Total CREB protein was shown as pan-CREB, and S133-phosphorylated CREB was shown as p-CREB. (j) Luminescence assays of H 2 O 2 production in BMDMs which were transfected by shRNA after TS or normal medium culture. (k) Illustration of mechanism for LAMP2a-PRDX1/CRTC1 axis.

Article Snippet: For bafilomycin A1 treatment, 10 nM final concentration of bafilomycin (InvivoGen) was added with TS in cultured medium.

Techniques: Activation Assay, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Transfection, Genetically Modified, Derivative Assay, Plasmid Preparation, Over Expression, Co-Culture Assay, shRNA

Journal: Cells

Article Title: Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

doi: 10.3390/cells11193018

Figure Lengend Snippet: Autophagy flux is induced in myotonic dystrophy type 1 (DM1). ( A – E ) CTRL and DM1 cells were treated with 1 µM rapamycin (RAPA) or 100 nM bafilomycin A1 (BAF.A1) for 2 h. Expression levels of LC3-II ( A , B , D , E ) and p-S6(Ser235/236) ( A , C ) were assessed by immunoblotting and their densitometry was normalized to the loading control, GAPDH. Data are the mean ± SD of two replicates (* p < 0.05, *** p < 0.001, **** p < 0.0001 versus untreated cells, ¤¤ p < 0.01 versus BAF.A1-treated CTRL cells, with two-way ANOVA-Tukey’s test), arbitrary units (a.u). Each group (CTRL or DM1) consisted of three cell lines. ( F , G ) CTRL and DM1 cells were treated with BAF.A1 for 4 h. ( F ) Cells were immunolabeled with SQSTM1 antibody (Red) and nuclei stained with DAPI (blue), scale bar represents 10 μm. The original magnification is ×60. G/Quantification of SQSTM1 dots per cell was determined using ImageJ software ( n = 100 cells/condition). Data are the mean ± SD of two replicates (** p < 0.01 versus untreated cells and ¤ p < 0.05 versus BAF.A1-treated CTRL cells, with two-way ANOVA-Tukey’s test). ( H ) Represents the SQSTM1 mRNA expression level determined by qPCR. Data are the mean ± SD of two replicates (* p < 0.05 respect to CTRL cells). Each group (CTRL or DM1) consisted of three cell lines. All experiments were performed at least three times.

Article Snippet: Cells were seeded in 6-well plates 24 h prior to any treatment with the following compounds: bafilomycin A1 (BAF.A1, LC Laboratories, Woburn, MA, USA, R-5000, B-1080, 100 nM) [ ]; rapamycin (RAPA, Fisher, Pittsburgh, PA, USA, BP2963.1, 1 μΜ) [ ], Hegf (E9644, 100 ng/Ml), pepstatin A (P5318, 50–100 μM), DMSO as a vehicle (dilution factor 1:1000), all from Sigma-Aldrich; and leupeptin (Enzo, ALX-260–009, Farmingdale, NY, USA, 50–100 μM).

Techniques: Expressing, Western Blot, Control, Immunolabeling, Staining, Software

Journal: Cells

Article Title: Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

doi: 10.3390/cells11193018

Figure Lengend Snippet: Autophagy and cell death are induced in DM1 fibroblasts. ( A – C ) Fibroblasts from patients with DM1 were treated with 100 nM bafilomycin A1 (BAF.A1) and/or 1 μM rapamycin (RAPA) for 2 h. Protein expression levels of LC3-II ( A , B ) and p -S6 (Ser235/236) ( A , C ) were determined by immunoblotting and their densitometry was normalized to the loading control, GAPDH. Data correspond to the mean ± SD of three independent lines (** p < 0.01, **** p < 0.0001 compared with untreated cells, and ¤ p < 0.05, ¤¤ p < 0.01 versus BAF.A1-treated cells with one-way ANOVA-Tukey’s test), arbitrary units (a.u). ( D , E ) CTRL and DM1 cells were treated with 1 µM RAPA for 24 h and then stained with annexin and propidium iodide (PI). The percentages of annexin-positive ( D ) or PI-positive ( E ) cells were detected by flow cytometry. Data are the mean % ± SEM of three replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to CTRL cells and ¤¤¤¤ p < 0.0001 versus DM1 cells with two-way ANOVA-Tukey’s test), N = 10,000 events. Each group (CTRL or DM1) consisted of three cell lines. All experiments were performed at least three times.

Article Snippet: Cells were seeded in 6-well plates 24 h prior to any treatment with the following compounds: bafilomycin A1 (BAF.A1, LC Laboratories, Woburn, MA, USA, R-5000, B-1080, 100 nM) [ ]; rapamycin (RAPA, Fisher, Pittsburgh, PA, USA, BP2963.1, 1 μΜ) [ ], Hegf (E9644, 100 ng/Ml), pepstatin A (P5318, 50–100 μM), DMSO as a vehicle (dilution factor 1:1000), all from Sigma-Aldrich; and leupeptin (Enzo, ALX-260–009, Farmingdale, NY, USA, 50–100 μM).

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

Journal: Cells

Article Title: Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

doi: 10.3390/cells11193018

Figure Lengend Snippet: EGF-induced EGFR is sorted into lysosomes. ( A – D ) Serum-starved cells were incubated with 100 ng/mL EGF for one hour. ( A ) Cells were co-labeled with EGFR and LAMP1 antibodies. Nuclei were stained with 300 nM DAPI (scale bar is 10 μm; original magnification is ×60). ( B ) The perinuclear distribution of EGFR that was determined by manual counting, n = 80–100 cells (* p < 0.05, ** p < 0.01 two-way ANOVA Tukey’s test). ( C ) The fluorescence intensity of EGFR represented as integrated density from ImageJ software (** p < 0.01, **** p < 0.0001 two-way ANOVA Tukey’s test). ( D ) Reflects the co-localization between EGFR/LAMP1 using Pearson’s coefficient (** p < 0.01, **** p < 0.0001 with two-way ANOVA). All experiments were performed at least two times. ( E , F ) Serum-starved cells (CTRL and DM1) were pre-treated for 60 min with 100 nM BAF.A1 and then stimulated with 100 ng/mL EGF for one hour. ( F ) Represents the level of EGF-induced EGFR degradation normalized to GAPDH. Data are the mean ± SD of two replicates, (* p < 0.05, with two-way ANOVA Tukey’s test).

Article Snippet: Cells were seeded in 6-well plates 24 h prior to any treatment with the following compounds: bafilomycin A1 (BAF.A1, LC Laboratories, Woburn, MA, USA, R-5000, B-1080, 100 nM) [ ]; rapamycin (RAPA, Fisher, Pittsburgh, PA, USA, BP2963.1, 1 μΜ) [ ], Hegf (E9644, 100 ng/Ml), pepstatin A (P5318, 50–100 μM), DMSO as a vehicle (dilution factor 1:1000), all from Sigma-Aldrich; and leupeptin (Enzo, ALX-260–009, Farmingdale, NY, USA, 50–100 μM).

Techniques: Incubation, Labeling, Staining, Fluorescence, Software

Journal: The Journal of Cell Biology

Article Title: Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

doi: 10.1083/jcb.201505108

Figure Lengend Snippet: COMMD9 deficiency leads to lysosomal degradation of Notch. (A) HEK293 cells were transfected with a Notch2 expression vector; siRNA was used to concurrently silence COMMD9 expression. Notch2 expression was monitored using antibodies that recognize the extracellular domain (Notch2-N) or an intracellular epitope (Notch2-C). Molecular mass markers (in kD) are shown. (B) HEK293 cells were transfected with a Jagged1 expression vector; siRNA was used to concurrently silence COMMD9 expression. Jagged1 expression was evaluated using Jagged1 or FLAG antibodies. Molecular mass markers (in kD) are shown. (C) Levels of Notch1 and Notch2 expression in two Commd9 -deleted cell lines (−/−) and their isogenic controls (F/F) were determined by immunoblotting. Molecular mass markers (in kD) are shown. (D) Using Commd9 F/F and −/− MEFs, total Notch1 and Notch2 levels (whole-cell lysates [WCLs]) were determined in by immunoblotting (left). In addition, surface levels of both proteins (plasma membrane [PM]) were also determined by biotinylation and immunoblotting (right). Molecular mass markers (in kD) are shown. (E) The reduced expression of Notch2 shown in is rescued by Bafilomycin A1 (BafA) treatment, an inhibitor of endolysosomal acidification that impairs lysosomal proteases. Molecular mass markers (in kD) are shown.

Article Snippet: Treatment with Bafilomycin A1 (Tocris) consisted of adding this reagent to the growth media (100 nM) for 24 h. Stimulation with plate-bound Jagged1 (R&D Systems) consisted of first applying the ligand to empty growth plates for 24 h (0.5 μg per well of a six-well plate).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Clinical Proteomics, Membrane

Journal: Molecular Oncology

Article Title: Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

doi: 10.1016/j.molonc.2013.11.002

Figure Lengend Snippet: Reduced Notch signaling in Drosophila imaginal disc upon pharmacological inhibition of V‐ATPase. (A–C) Single confocal sections of 3rd instar wing imaginal discs from larvae expressing the Notch signaling reporter E(spl)mβ‐LacZ that have been fed with the indicated drug. Discs are stained with anti‐βGal to detect expression of the Notch target. Compared with discs from mock‐fed animals (A), discs from animals fed with DAPT (B) and BafA1 (C) show a significant decrease of Beta‐Gal expression. (D) Quantitative RT‐PCR on mRNA extracted from 3rd instar wing imaginal discs from flies fed with drugs as indicated. E(spl)mβ‐LacZ and E(spl)m7 show a 30–40% decrease upon γ‐secretase inhibition and more than 60% with V‐ATPase inhibitor BafA1. (E–F) Single confocal section of discs not expressing (E) or expressing (F) Shrub:GFP under the control of RnGAL4, a wing pouch specific driver. (G–L) High magnification confocal sections of wing pouch cells not expressing (G, I, K) or expressing (H, J, L) Shrub:GFP. Discs have been stained to detect Ubiquitin (G–H), the endosomal marker Avl (I–J) or the Notch intracellular domain (NICD; K‐L). In discs expressing Shrub:GFP, accumulation of ubiquitinated cargoes, including Notch, at endosomal sites is observed. Single channels for Ubiquitin, Avl and NICD are shown in H′, J′, L′. (M) Quantitative RT‐PCR on mRNA extracted from wing imaginal discs from Shrub:GFP expressing flies fed with drugs and induced as indicated. E(spl)mβ expression is 30% reduced upon feeding with BafA1 and 50% upon γ‐secretase inhibition. Comparable GFP expression levels indicate equal amounts of Shrub:GFP expressing cells in induced samples under different drug treatment.

Article Snippet: Is it possible that in CCRF‐CEM cells BafA1 treatment affects Akt signaling downstream of phopho‐Akt?

Techniques: Inhibition, Expressing, Staining, Quantitative RT-PCR, Control, Ubiquitin Proteomics, Marker

Journal: Molecular Oncology

Article Title: Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

doi: 10.1016/j.molonc.2013.11.002

Figure Lengend Snippet: Impaired Notch‐signaling activity by V‐ATPase inhibition during vertebrate development. (A–E) Representative 28 hpf zebrafish embryos treated as at gastrulation as indicated and visualized in bright field, lateral view. Compared to mock‐treated embryo (A) embryos incubated with varying doses of DAPT (B–C) or BafA1 (D–E) or both (F) show mild morphological defects. (A′–F′) GFP levels associated to the expression of the Notch reporter line Tg(Tp1bglob:eGFP)ˆum14 visualized in the same embryos. Incubation with DAPT or BafA1 results in dose‐dependent reduction of GFP signal (B′–F′). Note that combination of both drugs at low dose leads to a reduction of GFP expression that is comparable to treatment with either of the drugs at high dose (compare B′ and D′ with F′).

Article Snippet: Is it possible that in CCRF‐CEM cells BafA1 treatment affects Akt signaling downstream of phopho‐Akt?

Techniques: Activity Assay, Inhibition, Incubation, Expressing

Journal: Molecular Oncology

Article Title: Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

doi: 10.1016/j.molonc.2013.11.002

Figure Lengend Snippet: V‐ATPase inhibition reduces Notch signaling activation in human breast cells. (A–D) Single confocal section showing the subcellular localization of endogenous Notch1 in MCF10A upon EGTA stimulation in cells treated as indicated. Cells have been stained with an anti‐Notch1, Phalloidin and DAPI were used to highlight the cell cortices and nuclei. Nuclear Notch1 localization is completely inhibited by DAPT treatment and is significantly reduced by BafA1 treatment. Single channels for anti‐Notch1 are shown in A′–D′. Nuclear pixel intensity quantification of panel A′–D′ is shown in E. (F) Western blot analysis of full length Notch1 (300 KDa) and cleaved Notch1 (cNICD1) on extracts from MCF10A treated as indicated. Stimulation with EGTA leads to production of the γ‐secretase‐cleaved active form of Notch (cNICD1), which is reduced upon pretreatment with BafA1 and abolished upon pretreatment with DAPT. (G) Quantitative RT‐PCR to detect Hes1 expression levels in MCF10A cells. Cells were pretreated with DMSO, DAPT, or BafA1 and stimulated with EGTA. Upon BafA1 pretreatment we observe a 20% reduction of Hes1 expression.

Article Snippet: Is it possible that in CCRF‐CEM cells BafA1 treatment affects Akt signaling downstream of phopho‐Akt?

Techniques: Inhibition, Activation Assay, Staining, Western Blot, Quantitative RT-PCR, Expressing

Journal: Molecular Oncology

Article Title: Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

doi: 10.1016/j.molonc.2013.11.002

Figure Lengend Snippet: V‐ATPase inhibition impairs Notch signaling and associated growth in breast cancer cells. (A–D) Cell culture growth after 7 days upon treatment as indicated. Growth of normal breast MCF10A cells and breast cancer HCC1187 cells, which harbor a translocation leading to expression of a cytoplasmic active Notch truncation, is not affected by drug treatments. In contrast, growth of breast cancer HCC2218 and HCC1599 cells, which harbor translocations leading to expression of a membrane tethered, active Notch truncation, is sensitive to both DAPT and BafA1 in a dose‐sensitive fashion. Note that combination of both drugs at low dose in HCC2218 cells results in a growth inhibition that is comparable to treatment with either of the drugs at high dose. (E–F) Western blot analysis of cleaved Notch1 (cNICD1) on extracts from HCC1599 and HCC2218 treated for 7 days as indicated. cNICD1 production is strongly reduced upon pretreatment with BafA1 and DAPT. (G) Quantitative RT‐PCR to detect Hes1 expression levels in HCC1599 cells treated as indicated. Upon BafA1 pretreatment we observe a 25% reduction of Hes1 expression.

Article Snippet: Is it possible that in CCRF‐CEM cells BafA1 treatment affects Akt signaling downstream of phopho‐Akt?

Techniques: Inhibition, Cell Culture, Translocation Assay, Expressing, Membrane, Western Blot, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

doi: 10.1016/j.molonc.2013.11.002

Figure Lengend Snippet: V‐ATPase inhibition reduces growth of T‐cell leukemia cell lines. (A) Cell culture growth after 7 days upon treatment as indicated. Growth of T‐cell leukemia DND‐41 cells, which harbor activating Notch1 mutations, is sensitive to both DAPT and BafA1 in a dose‐sensitive fashion. Note that combination of both drugs at low dose results in a growth inhibition that is comparable to treatment with either of the drugs at high dose. (B–C) Western blot analysis of cNICD1 (B) or of Akt and pAKT S473 (C) on extracts from DND‐41 cells treated as indicated. cNICD1 production is not reduced upon pretreatment with BafA1. In contrast, pAKT S473 levels are reduced upon pretreatment with BafA1 and DAPT. (D–E) Cell culture growth after 7 days upon treatment as indicated. Growth of T‐cell leukemia DND‐41 cells (D) and of CCRF‐CEM cells (E), which also harbor inactivating PTEN mutations, is sensitive to the Akt signaling inhibitor SH6, but not to GSIs. Note that combination of SH6 with BafA1 results in maximal growth inhibition. (F–G) Western blot analysis of cNICD1 (F) or of Akt and pAKT S473 (G) on extracts from CCRF‐CEM cells treated as indicated. cNICD1 production and pAKT levels are not reduced upon pretreatment with BafA1.

Article Snippet: Is it possible that in CCRF‐CEM cells BafA1 treatment affects Akt signaling downstream of phopho‐Akt?

Techniques: Inhibition, Cell Culture, Western Blot