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: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a – d Representative western blot (WB) images of ACBP, SQSTM1, LC3-II/LC3-I, GAPDH and Stain-free gel levels ( a ) with quantifications for ACBP ( b ), SQSTM1 ( c ), and the LC3-II/LC3-I interconversion ratio ( d ). n = 9 mice per group (biological replicates). e – g Representative WB images of ACBP, SQSTM1 and GAPDH ( e ) with respective quantifications for ACBP ( f ) and autophagy flux calculation as the ratio SQSTM1(Leu)/SQSTM1(Unt) ( g ). n = 3 mice per group. h , i ACBP levels in plasma in mice aged 6- or 20-week-old, WT or Alms1 -/- ( h ), under ad libitum feeding conditions or after 24 h period of caloric restriction (CR) ( i ). n = 5 mice (WT and Alms1 -/- ad libitum ), n = 8 mice ( Alms1 WT, CR) and). n = 10 mice ( Alms1 -/- , CR) per group. j , k Representative WB images of FASN, SQSTM1, ACBP and GAPDH ( j ) in primary hepatocytes isolated from mice WT or Alms1 -/- and respective quantifications ( k ). n = 3 mice per group (biological replicates). l , m Representative WB images of ALMS1, ACBP and GAPDH ( l ) in HepG2 cells silenced (knockdown, KD) with an unrelated control (UNR) or two specific siRNA sequences targeting ALMS1 , ± bafilomycin A1 (BafA1) for 4 h ( m ). n = 4 independent experiments. n – p PCR verification of ALMS1 knockout (KO) in HepG2 cells or their wild-type (WT) control ( n ) and representative western blot images of ACBP and GAPDH ( o ) in 2 clones for ALMS1 KO HepG2 cell line, or respective control WT, ± BafA1 for 4 h ( p ). n = 3 independent experiments. All WB quantifications were normalized using GAPDH as a loading control. The data are represented as the mean ± SEM, with individual mouse/replicate values depicted as circles, and exact P- values are shown on the figure. Source data are provided as a Source Data file. P- values were calculated using two-sided unpaired t tests ( b – d , g – h , k ). For panels with multiple comparisons, P- values were adjusted (two-sided) using the Sidak method ( f , i ) or Tukey’s test ( m , p ). Abbreviations used: Bp base pair; kDa kilodalton; MW molecular weight; r.u .: relative units; Unr unrelated; empty control vector for gene silencing as a reference.

Article Snippet: Cells were seeded in 6–12-well plates and grown for 36 h before treatment with 50 nM BafA1 (S-S1413, Selleckchem), EBSS medium (24010043, Gibco) or anti-ACBP (or respective isotype control) in vitro (1–10 μg/mL; ab232760, Abcam).

Techniques: Western Blot, Staining, Clinical Proteomics, Isolation, Knockdown, Control, Knock-Out, Clone Assay, Molecular Weight, Plasmid Preparation

Journal: Life Science Alliance

Article Title: Endolysosome-targeted nanoparticle delivery of antiviral therapy for coronavirus infections

doi: 10.26508/lsa.202403182

Figure Lengend Snippet: (A) General pipeline outlining FlowJo gating of viable, single cells using forward and side scattering, resulting in histogram frequency distribution of individual cell fluorescence. Positive cell gating is based on unstained and untreated controls; representative unstained and stained controls from the DQ-Red BSA assay are shown. (B, C) Rho-NP uptake measured by flow cytometry in Vero E6 cells and displayed as (B) representative intensity histogram or (C) median intensity bar graph. (D, E) Rho-NP uptake measured by flow cytometry in HFL1 cells and displayed as (D) representative intensity histogram or (E) median intensity bar graph. (F) Representative images of Vero E6 cells treated with NPs ± MFQ (100 μg/ml), free MFQ (20 μM), bafilomycin A1 (200 nM), or control and stained with Hoechst 33342 (blue) and LysoTracker Green (green) to probe for lysosome accumulation. Scale bars = 20 μm. (G) Quantification of lysosomal accumulation. Statistical significance was determined by one-way ANOVA: ** P < 0.01, *** P < 0.001, **** P < 0.0001 against untreated controls. All experiments represent N = 3 biological replicates, which represent the mean of at least n = 3 technical replicates. The intensity histogram displays a single representative cell population per timepoint. The intensity bar graph is displayed as medians ±SEM. All other experiments are displayed as means ± SEM. Source data are available for this figure.

Article Snippet: HFL1 cells were cultured in a 96-well plate at 15,000 cells/well for 24 h, after which the media were exchanged for media containing no-treatment control, empty NPs (12.5, 25, 50, 75, 100 μg/ml NPs), or MFQ-NPs (12.5, 25, 50, 75, 100 μg/ml NPs) for 24 h. Control treatments include bafilomycin A1 (200 nM), and Pepstatin A (10 μg/ml) + E64d (10 μg/ml) for 4 h, or free MFQ (10, 15 μM) for 24 h. After treatment, cells were washed with 1X PBS and incubated with 10 μg/ml DQ-Red BSA reagent (Thermo Fisher Scientific) in culture media for 1 h. After incubation with assay reagent, cells were trypsinized, washed with 1X PBS by centrifugation, resuspended in FACS buffer (PBS + 2% FBS), and then subjected to flow cytometry.

Techniques: Fluorescence, Staining, Flow Cytometry, Control

Journal: Life Science Alliance

Article Title: Endolysosome-targeted nanoparticle delivery of antiviral therapy for coronavirus infections

doi: 10.26508/lsa.202403182

Figure Lengend Snippet: (A) Representative confocal images of lysosomal pH measurements using LysoSensor Yellow/Blue dextran in Vero E6 cells treated with NP (±MFQ) 100 μg/ml, free MFQ 10 μM, bafilomycin 200 nM, or control. Scale bars = 10 μm. (B) Quantification of lysosomal pH. (C) Representative images of Calu-3 cells treated with NPs (±MFQ) 100 μg/ml, free MFQ 20 μM, bafilomycin 200 nM, or control and stained with Hoechst 33342 (blue) and LysoTracker Green (green) to probe for lysosome accumulation. Scale bars = 20 μm. (D) Quantification of lysosomal accumulation. (E) Quantification of lysosomal protease activity by DQ-Red BSA assay in Vero E6 cells treated with NPs (±MFQ), free MFQ, bafilomycin A1, pepstatin A + E64d, or controls at the indicated concentrations. Statistical significance was determined by one-way ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 against untreated controls. Each independent LysoSensor imaging experiment represents the mean of n = 10–20 individual images. All other biological replicates represent the mean of n = 3 technical replicates. All experiments represent N = 3 biological replicates. All data are displayed as means ± SEM. Source data are available for this figure.

Article Snippet: HFL1 cells were cultured in a 96-well plate at 15,000 cells/well for 24 h, after which the media were exchanged for media containing no-treatment control, empty NPs (12.5, 25, 50, 75, 100 μg/ml NPs), or MFQ-NPs (12.5, 25, 50, 75, 100 μg/ml NPs) for 24 h. Control treatments include bafilomycin A1 (200 nM), and Pepstatin A (10 μg/ml) + E64d (10 μg/ml) for 4 h, or free MFQ (10, 15 μM) for 24 h. After treatment, cells were washed with 1X PBS and incubated with 10 μg/ml DQ-Red BSA reagent (Thermo Fisher Scientific) in culture media for 1 h. After incubation with assay reagent, cells were trypsinized, washed with 1X PBS by centrifugation, resuspended in FACS buffer (PBS + 2% FBS), and then subjected to flow cytometry.

Techniques: Control, Staining, Activity Assay, Imaging

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

Journal: Stem Cell Reports

Article Title: Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

doi: 10.1016/j.stemcr.2014.03.014

Figure Lengend Snippet: Genetic Correction of Autophagy Phenotype in NPC1 iPSC-Derived cells (A and B) Immunoblot analysis and quantifications of p62 and LC3-II levels using anti-p62, anti-LC3, and anti-GAPDH antibodies in hepatic (A) and neuronal cultures (B) derived from indicated control and NPC1 iPSC lines. Graphical data represent mean ± SE (n = 4). (C and D) Immunoblot analysis and quantification of LC3-II levels using anti-LC3 and anti-GAPDH antibodies in hepatic (C) and neuronal cultures (D) derived from control and NPC1 iPSC lines, treated with or without 400 nM bafilomycin A 1 (BafA 1 ) for 4 hr. Graphical data represent mean ± SE (n = 3). (E and F) Immunoblot analysis and quantifications of p62 and LC3-II levels in hepatic (E) and neuronal cultures (F) derived from the NPC1-2 iPSC line after correction of the NPC1 I1061T mutation. Graphical data represent mean ± SE (n = 3). (G) Electron microscopy images of representative NPC1 iPSC-derived hepatic-like cells before and after genome editing. Arrows are indicating autophagic vacuoles. Graphical data represent mean ± SE (n = 3). Nucleus (N), rough endoplasmatic reticulum (rER), mitochondria (M), Golgi (G). Scale bar, 500 nm. Results shown are representative for at least three independent experiments using two different clones of each line. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, nonsignificant.

Article Snippet: Compounds used for treatment of hepatic-like cells were bafilomycin A1 (Enzo Life Sciences), rapamycin (LC Laboratories), carbamazepine, verapamil, trehalose, SMER28, and HP-β-cyclodextrin (all Sigma-Aldrich).

Techniques: Derivative Assay, Western Blot, Mutagenesis, Electron Microscopy, Clone Assay

Journal: Translational Neuroscience

Article Title: CST3 alleviates bilirubin-induced neurocytes’ damage by promoting autophagy

doi: 10.1515/tnsci-2022-0314

Figure Lengend Snippet: CST3 protects HT22 cells from UCB damage via promoting autophagy: (a) CST3 promotes HT22 cells autophagy, (b) mTOR, (c) pmTOR, (d) Beclin1, (e) LC3II/LC3I, (f) viability of HT22 cells treated with CST3 and autophagy inhibitor Bafilomycin A1, and (g) viability of HT22 cells treated with CST3 and autophagy activator rapamycin. HBR: high-dose UCB group; +: adding or overexpression; -: not adding; --: knockdown. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: We performed MTT (Solarbio, China) assays to examine the viability of HT22 cells treated with CST3 (100 pg/μL), UCB (170 μM), or bafilomycin A1 (200 nM and added during the last 3 h of treatment).

Techniques: Over Expression, Knockdown