Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: HDAC inhibition reduces Notch3 levels and signaling in T-ALL cells. a T-ALL cells (DND 41, MOLT3, and Jurkat) were treated with TSA (0.5 µM) or solvent (DMSO) for 16 h and protein levels analyzed by western blot. Actin was used as a loading control and tubulin acetylation and c-Myb levels as markers of HDAC inhibition. b TSA reduces Notch3 surface expression in T-ALL cells. DND 41 and MOLT3 cells treated with TSA or DMSO for 16 h were stained with PE anti-human Notch3 (anti-N3 Ab) or with isotype control antibody and analyzed by flow cytometry. One representative experiment of three performed is shown. Histogram reports fluorescence mean intensity (FMI) ± SD of three independent experiments (** P < 0.01; * P < 0.05). c TSA reduces Notch3 expression in PDX-derived T-ALL cells. T-ALL cells obtained from the spleen of xenografted mice were treated in vitro with TSA for 16 h and protein levels were analyzed by western blot. d-h Effects of TSA on Notch3 target genes and on Notch transcript levels. T-ALL samples, including both c ell lines ( d, f, g ) and PDX T-ALL cells ( e , h ), were treated with TSA or Givinostat (GIV) (2 µM) for 16 h and mRNA levels of NOTCH3, c-MYB , or Notch target transcripts ( pTα, CR2, DTX-1 ) were analyzed by qRT-PCR. n.d.: not detectable. Statistically significant differences are indicated (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments). Expression data are normalized to DMSO samples

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, Solvent, Western Blot, Control, Expressing, Staining, Flow Cytometry, Fluorescence, Derivative Assay, In Vitro, Quantitative RT-PCR

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: HDAC inhibition promotes Notch3 degradation through the lysosomal pathway. a MOLT3 cells were treated with cyclohexymide (CHX, 500 µM) or with CHX plus TSA (0.5 µM). At 1, 5, 8, and 16 h, protein levels of c-Myb and Notch3 FL were analyzed. One representative western blot is reported. b c-Myb (left) and Notch3 FL (right) protein expression in three independent experiments was measured by densitometric analysis and normalized to Actin (** P < 0.01). MOLT3 ( c ) or TALL1 cell lines ( d ) were treated with TSA plus MG132 (20 µM) or chloroquine (CHL) (20 µM) for 16 h followed by western blot analysis. Numbers indicate results of densitometric analysis of Notch3 FL bands normalized to Actin. ( e ) MOLT3 were pre-treated with ciliobrevin D (20 µM) for 24 h and then with vehicle or TSA (0.5 µM) for 16 h. Cells were stained with PE anti-N3 antibody and analyzed by FACS analysis. Statistically significant differences are indicated (** P < 0.01, mean ± SD of three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, Western Blot, Expressing, Staining

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: TSA increases co-localization of Notch3 protein with LAMP2-positive vesicles and increases the abundance of Notch3 FL in the lysosomal compartment. MOLT3 cells were treated with DMSO or TSA (0.5 µM) for 8 h and were subsequently immuno-stained for Notch3 and LAMP2. n = 21 DMSO-treated cells and n = 24 TSA-treated cells were analyzed by z-stack laser scanning microscopy using a ×63 oil objective. Images were acquired with a resolution of 1024 × 1024 pixels. a, b Representative optical slices taken in the apical portion of the cells above the nucleus are shown. Scatterplots on the right-hand portion of the panels indicate the fluorescence intensity of Notch3 ( X- axis) and LAMP2 ( Y- axis) detected in each pixel. Detection thresholds were set at 1000 for both channels. Region 1: Notch3 single positive pixels. Region 2: LAMP2 single positive pixels. Region 3 double-positive (Notch3/LAMP2) pixels. c Co-localization coefficients were calculated in 21 mock-treated cells and 24 TSA-treated cells using the Zeiss Histogram software tool. At least 15 optical slices were analyzed for each cell. The values of the co-localization coefficients range between 0 and 1. Box plots reported medians, lower/upper quartiles, and outliers of all co-localization coefficients obtained for DMSO and TSA cells respectively. *** indicates P < 0.001 (Mann–Whitney test). d DND 41 cells were treated with DMSO or TSA (0.5 µM) for 8 h and 1 × 10 8 pelleted cells underwent subcellular fractionation. Protein extracts for each fraction were analyzed by western blot. A representative blot is reported. WCL whole-cell lysate; L lysosome; PM plasma membrane. e Histogram reports the quantified ratio of lysosomal enriched Notch3 FL normalized to Notch3 FL present on the plasma membrane. Expression of Notch3 FL in the different fractions was normalized to each fraction-specific proteins. Mean ± SD of two independent replicates (* P < 0.05, Student’s t -test)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Staining, Laser-Scanning Microscopy, Fluorescence, Software, MANN-WHITNEY, Fractionation, Western Blot, Clinical Proteomics, Membrane, Expressing

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: Pharmacologic inhibition of HDAC6 lowers Notch3 FL protein levels and signaling, triggering apoptosis of T-ALL cells. a T-ALL cell lines (up) and PDX T-ALL cells (bottom) were treated in vitro for 16 h with the HDAC6 inhibitor tubacin (TUB, 2 µM), HDAC1 inhibitor (HDAC1i, 2 µM), or HDAC8 inhibitor (HDAC8i, 2 µM). Protein levels were analyzed by western blot. b Tubacin reduces Notch3 surface expression in T-ALL cells. DND 41 and MOLT3 cells treated with tubacin (2 µM) or DMSO for 16 h were stained with PE anti-human Notch3 (anti-N3 ab) and analyzed by flow cytometry. One representative experiment of three performed is shown. Histogram reports fluorescence mean intensity (FMI) ± SD of three independent experiments (*** P < 0.001). Expression levels of NOTCH3 ( c ) and Notch target genes ( d ) in T-ALL cells treated as above were analyzed by qRT-PCR (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments). Expression data are normalized to DMSO samples. Induction of apoptosis by HDACi in T-ALL cells ( e ) and in PDX cells ( f ) was measured by flow cytometric analysis of Annexin V staining (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Staining, Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: Effects of HDAC6 silencing on Notch3 FL protein levels and apoptosis of T-ALL cells. MOLT3 (top), TALL1 (middle), or SUPT11 (bottom) cells were transduced with lentiviral vectors encoding a scramble shRNA or two different shHDAC6 vectors. qRT-PCR ( a ) and western blot analysis ( b ) performed 96 h after transduction, confirmed the efficacy of HDAC6 silencing by both constructs. HDAC6 mRNA levels were normalized setting at one the shRNA sample. c MOLT3 or TALL1 cells expressing shHDAC6 #1 or shHDAC6 #2 showed reduced Notch3 FL protein levels. Western blot analysis was not performed in SUPT11 cells as it is known that these cells do not express detectable Notch3 . d HDAC6 silencing was associated with increased apoptosis of T-ALL cells which express Notch3, but not in SUPT11 cells. Apoptosis was analyzed by caspase 3–7 assay 5 days after transduction of cells with the indicated LV. Relative Luminescence Units (RLU) are reported (* P < 0.05; ** P < 0.01, *** P < 0.001, mean ± SD, three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Transduction, shRNA, Quantitative RT-PCR, Western Blot, Construct, Expressing

Journal: Blood Advances

Article Title: Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

doi: 10.1182/bloodadvances.2020002666

Figure Lengend Snippet: AML cells have high levels of constitutive autophagy, compared with normal CD34+cells and are sensitive to autophagy inhibition. Immunoblot analysis of the indicated AML cell lines (A) and patient samples (B) of LC3 and actin (loading control) showed different levels of basal autophagy. Quantification of LC3-I and -II levels was performed with Image J software. (C) The KG-1 and HL60 cell lines were incubated with NH4Cl overnight (>12 hours), and the lysates were analyzed for LC3 and actin (loading control). Increased LC3-II after treatment indicates active autophagic flux. (D) CD34+ cells from CB, BM, or patients with AML were stained with antibodies against LC3 (green) as a marker of autophagosomes or LAMP-2 (red) as a marker of lysosomes with DAPI (blue) as a nuclear stain. (E) Electron micrograph images show more autophagosomes in AML cells, compared with nonmalignant CB cells. Original magnification ×15 000, ×30 000 zoom. (F) Samples from AML patients were treated with increasing concentrations of autophagy inhibitors CQ, 3-MA, or Baf A1. Cell viability was measured with YOPRO-1 and 7-AAD and is displayed as the percentage of viable cells, compared with the untreated controls.

Article Snippet: Human AML cell lines (HL60 and MOLM-13) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Western Blot, Control, Software, Incubation, Staining, Marker

Journal: Blood Advances

Article Title: Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

doi: 10.1182/bloodadvances.2020002666

Figure Lengend Snippet: Baf A1 preferentially targets functionally defined LSCs and leads to the accumulation of damaged mitochondria. (A) Primary cells from patients with AML were grown in methylcellulose with the indicated drug (7.5 µM PTL, 2.5 µM Ara-C, 40 µM CQ, 50 nM Baf A1, and 10 mM 3-MA). Data are shown as the number of CFUs, compared with those in untreated cells. After the primary plating was quantified, the cells were collected for a secondary replating (technical replicates). Error bars represent ± standard error of the mean (SEM). Statistical analysis compared each treatment mean with that of Baf A1. (B) NSG mice were inoculated with samples from 2 patients with AML (AML 1 and AML 2) and treated with DMSO or Baf A1 (1 mg/kg) intraperitoneally 2 times per week for 4 weeks (5 mice per group). The BM was then harvested from the mouse recipients of the primary transplant, measured for AML burden by flow cytometry for human and mouse CD45, and injected via the tail vein into the second set of NSG mice. The AML burden of the secondary transplant was measured after 6 to 8 weeks. (C) Electron micrographs of healthy donor CB or AML18 samples after overnight treatment with Baf A1 (25 nM). AML cells demonstrated an increase in the number of mitochondria. Original magnification ×15 000. (D) CD34+-enriched samples from patients with AML were treated with 25 nM Baf A1 for 48 hours, then stained for mitochondria using MitoID (2 technical replicates). Data are shown as mean fluorescence intensity (MFI) of MitoID. (E-G) AML samples or MOLM-13 cells were treated with Baf A1 or CQ for 48 hours. Viability of all samples was >85%, by trypan blue exclusion. (E) Representative MitoStress Test profile determined with a Seahorse XF analyzer. (F) MitoID MFI (≥3 biological replicates; 3 technical replicates). (G) Average maximal respiration (≥3 biological replicates; 3 technical replicates). Error bars represent ± standard deviation. *P < .05; ** P < .01; *** P < .001. ns, not significant.

Article Snippet: Human AML cell lines (HL60 and MOLM-13) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Injection, Staining, Fluorescence, Standard Deviation

Journal: Blood Advances

Article Title: Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

doi: 10.1182/bloodadvances.2020002666

Figure Lengend Snippet: Hypoxia induces autophagic flux in AML cell lines. AML cell lines were incubated in normoxia or hypoxia for 48 hours, with or without lysosomal protease inhibitors (10 µg/mL pepstatin A and E-64d) added for the final 6 hours. (A) Immunoblot analysis of LC3 and actin (loading control) is shown. LC3-II levels were determined by densitometric analysis and normalized to actin. Values are relative to untreated cells in 21% O2. (B) Immunoblot analysis of p62 and actin (loading control). Blots are representative of 2 independent experiments. (C) HEL-Luc cells were stained with Cyto-ID, fixed, mounted in Vectashield+DAPI, and imaged on a Leica TCS SP2 spectral confocal microscope with a 63× objective (blue, nucleus/DAPI; green, autophagosomes/Cyto-ID). Bar represents 10 µm. (D) Microscopic images were quantified by ImageJ to determine the percentage of Cyto-ID+ cells (3 biological replicates, ≥100 cells counted per replicate). Error bars represent ± standard deviation. *P < .05.

Article Snippet: Human AML cell lines (HL60 and MOLM-13) were purchased from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Control, Staining, Microscopy, Standard Deviation

Journal: Blood Advances

Article Title: Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

doi: 10.1182/bloodadvances.2020002666

Figure Lengend Snippet: Sensitivity to autophagy inhibitors in hypoxia correlates with the extent of inhibition of mitochondrial respiration. (A-B) MOLM-13 AML cell lines were treated with the indicated concentrations of CQ (A) or Baf A1 (B) in normoxia or hypoxia for 72 hours and analyzed by annexin V/PI flow cytometry (≥3 biological replicates; 2 technical replicates). (C-D) MOLM-13 cells were transfected with siRNAs against Atg-5 and Atg-7. After 24 hours, they were placed in normoxia or hypoxia. Lysates harvested after 48 hours for immunoblot analysis (C) (quantification under each blot normalized to actin, followed by normalization to NT in normoxia), or the cells were analyzed after 72 hours by annexin V/PI flow cytometry (D) (3 biological replicates; 2 technical replicates). (E-F) MOLM-13 cells were treated with CQ (E) or Baf A1 (F) for 48 hours in normoxia or hypoxia and analyzed on a Seahorse XF analyzer. Representative MitoStress Test profiles and average maximal respiration for cells are shown (2 biological replicates; 3 technical replicates). (G-H) MOLM-13 cells were treated with CQ (G) or Baf A1 (H) for 48 hours, stained with MitoID, and analyzed by flow cytometry (3 biological replicates; 2 technical replicates). Data are shown as average mean fluorescence intensity (MFI). (I) MOLM-13 cells were harvested after a 48-hour treatment with 10 µM CCCP, 50 µM CQ, or 25 nM Baf A1 and blotted for PINK-1. The levels were determined by densitometric analysis and normalized to actin. Values are shown relative to DMSO. (J) MOLM-13 cells were treated with 10 µM CCCP or 50 µM CQ, alone or in combination in normoxia or hypoxia for 72 hours, and analyzed by annexin V/PI flow cytometry (3 biological replicates; 2 technical replicates). Error bars represent ± standard deviation. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Article Snippet: Human AML cell lines (HL60 and MOLM-13) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Flow Cytometry, Transfection, Western Blot, Staining, Fluorescence, Standard Deviation

Journal: Blood Advances

Article Title: Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

doi: 10.1182/bloodadvances.2020002666

Figure Lengend Snippet: Hypoxia reduces chemosensitivity in AML cell lines, which can be restored with autophagy inhibition. The indicated AML cell lines were treated with Ara-C (100 nM HEL-Luc, 250 nM HL60, and MOLM-13 in normoxia (21% O2) or hypoxia (1% O2). Concentrations were determined based on cell line sensitivity to Ara-C. (A) Cells were processed after 72 hours for measurement of apoptosis by flow cytometry with annexin V/7-AAD or PI staining. Data are shown as a percentage of annexin V– and/or 7-AAD– or PI-positive cells normalized to untreated cells (≥3 biological replicates; 2 technical replicates). (B) HEL-Luc cell lines were treated with 100 nM Ara-C for 72 hours, and the total number of cells was quantified (4 biological replicates). (C) HEL-Luc cell lines were treated with 100 nM Ara-C, and phospho-H2A.X flow cytometry was performed after 48 hours. Data are shown as the MFI of p-H2A.X staining normalized to untreated in normoxia (2 biological replicates). (D-E) MOLM-13 and HL60 cells were treated with 250 nM Ara-C in normoxia (21% O2) or hypoxia (1% O2) in combination with bafilomycin A1 (Baf A1; 2 nM) (D) or chloroquine (CQ; 25 µM) (E). The cells were processed after 72 hours for apoptosis flow cytometry, using annexin V/7-AAD or PI staining. Data are shown as a percentage of annexin V– and/or 7-AAD– or PI-positive cells (3 biological replicates; 2 technical replicates). Error bars represent ± standard deviation. *P < .05; **P < .01; ***P < .001.

Article Snippet: Human AML cell lines (HL60 and MOLM-13) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Flow Cytometry, Staining, Standard Deviation

Journal: Cancer cell

Article Title: Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy

doi: 10.1016/j.ccell.2017.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: MOLM-13 , DSMZ , Cat#ACC-554; RRID: CVCL_2119.

Techniques: Recombinant, Imaging, Staining, Mutagenesis, Transfection, shRNA, Negative Control, Plasmid Preparation, Control, Software, In Vivo Imaging, Microscopy, Flow Cytometry

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: HDAC inhibition reduces Notch3 levels and signaling in T-ALL cells. a T-ALL cells (DND 41, MOLT3, and Jurkat) were treated with TSA (0.5 µM) or solvent (DMSO) for 16 h and protein levels analyzed by western blot. Actin was used as a loading control and tubulin acetylation and c-Myb levels as markers of HDAC inhibition. b TSA reduces Notch3 surface expression in T-ALL cells. DND 41 and MOLT3 cells treated with TSA or DMSO for 16 h were stained with PE anti-human Notch3 (anti-N3 Ab) or with isotype control antibody and analyzed by flow cytometry. One representative experiment of three performed is shown. Histogram reports fluorescence mean intensity (FMI) ± SD of three independent experiments (** P < 0.01; * P < 0.05). c TSA reduces Notch3 expression in PDX-derived T-ALL cells. T-ALL cells obtained from the spleen of xenografted mice were treated in vitro with TSA for 16 h and protein levels were analyzed by western blot. d-h Effects of TSA on Notch3 target genes and on Notch transcript levels. T-ALL samples, including both c ell lines ( d, f, g ) and PDX T-ALL cells ( e , h ), were treated with TSA or Givinostat (GIV) (2 µM) for 16 h and mRNA levels of NOTCH3, c-MYB , or Notch target transcripts ( pTα, CR2, DTX-1 ) were analyzed by qRT-PCR. n.d.: not detectable. Statistically significant differences are indicated (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments). Expression data are normalized to DMSO samples

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, Solvent, Western Blot, Control, Expressing, Staining, Flow Cytometry, Fluorescence, Derivative Assay, In Vitro, Quantitative RT-PCR

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: HDAC inhibition promotes Notch3 degradation through the lysosomal pathway. a MOLT3 cells were treated with cyclohexymide (CHX, 500 µM) or with CHX plus TSA (0.5 µM). At 1, 5, 8, and 16 h, protein levels of c-Myb and Notch3 FL were analyzed. One representative western blot is reported. b c-Myb (left) and Notch3 FL (right) protein expression in three independent experiments was measured by densitometric analysis and normalized to Actin (** P < 0.01). MOLT3 ( c ) or TALL1 cell lines ( d ) were treated with TSA plus MG132 (20 µM) or chloroquine (CHL) (20 µM) for 16 h followed by western blot analysis. Numbers indicate results of densitometric analysis of Notch3 FL bands normalized to Actin. ( e ) MOLT3 were pre-treated with ciliobrevin D (20 µM) for 24 h and then with vehicle or TSA (0.5 µM) for 16 h. Cells were stained with PE anti-N3 antibody and analyzed by FACS analysis. Statistically significant differences are indicated (** P < 0.01, mean ± SD of three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, Western Blot, Expressing, Staining

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: TSA increases co-localization of Notch3 protein with LAMP2-positive vesicles and increases the abundance of Notch3 FL in the lysosomal compartment. MOLT3 cells were treated with DMSO or TSA (0.5 µM) for 8 h and were subsequently immuno-stained for Notch3 and LAMP2. n = 21 DMSO-treated cells and n = 24 TSA-treated cells were analyzed by z-stack laser scanning microscopy using a ×63 oil objective. Images were acquired with a resolution of 1024 × 1024 pixels. a, b Representative optical slices taken in the apical portion of the cells above the nucleus are shown. Scatterplots on the right-hand portion of the panels indicate the fluorescence intensity of Notch3 ( X- axis) and LAMP2 ( Y- axis) detected in each pixel. Detection thresholds were set at 1000 for both channels. Region 1: Notch3 single positive pixels. Region 2: LAMP2 single positive pixels. Region 3 double-positive (Notch3/LAMP2) pixels. c Co-localization coefficients were calculated in 21 mock-treated cells and 24 TSA-treated cells using the Zeiss Histogram software tool. At least 15 optical slices were analyzed for each cell. The values of the co-localization coefficients range between 0 and 1. Box plots reported medians, lower/upper quartiles, and outliers of all co-localization coefficients obtained for DMSO and TSA cells respectively. *** indicates P < 0.001 (Mann–Whitney test). d DND 41 cells were treated with DMSO or TSA (0.5 µM) for 8 h and 1 × 10 8 pelleted cells underwent subcellular fractionation. Protein extracts for each fraction were analyzed by western blot. A representative blot is reported. WCL whole-cell lysate; L lysosome; PM plasma membrane. e Histogram reports the quantified ratio of lysosomal enriched Notch3 FL normalized to Notch3 FL present on the plasma membrane. Expression of Notch3 FL in the different fractions was normalized to each fraction-specific proteins. Mean ± SD of two independent replicates (* P < 0.05, Student’s t -test)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Staining, Laser-Scanning Microscopy, Fluorescence, Software, MANN-WHITNEY, Fractionation, Western Blot, Clinical Proteomics, Membrane, Expressing

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: Pharmacologic inhibition of HDAC6 lowers Notch3 FL protein levels and signaling, triggering apoptosis of T-ALL cells. a T-ALL cell lines (up) and PDX T-ALL cells (bottom) were treated in vitro for 16 h with the HDAC6 inhibitor tubacin (TUB, 2 µM), HDAC1 inhibitor (HDAC1i, 2 µM), or HDAC8 inhibitor (HDAC8i, 2 µM). Protein levels were analyzed by western blot. b Tubacin reduces Notch3 surface expression in T-ALL cells. DND 41 and MOLT3 cells treated with tubacin (2 µM) or DMSO for 16 h were stained with PE anti-human Notch3 (anti-N3 ab) and analyzed by flow cytometry. One representative experiment of three performed is shown. Histogram reports fluorescence mean intensity (FMI) ± SD of three independent experiments (*** P < 0.001). Expression levels of NOTCH3 ( c ) and Notch target genes ( d ) in T-ALL cells treated as above were analyzed by qRT-PCR (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments). Expression data are normalized to DMSO samples. Induction of apoptosis by HDACi in T-ALL cells ( e ) and in PDX cells ( f ) was measured by flow cytometric analysis of Annexin V staining (* P < 0.05, ** P < 0.01, *** P < 0.001, mean ± SD of three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Staining, Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: Oncogene

Article Title: Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

doi: 10.1038/s41388-018-0234-z

Figure Lengend Snippet: Effects of HDAC6 silencing on Notch3 FL protein levels and apoptosis of T-ALL cells. MOLT3 (top), TALL1 (middle), or SUPT11 (bottom) cells were transduced with lentiviral vectors encoding a scramble shRNA or two different shHDAC6 vectors. qRT-PCR ( a ) and western blot analysis ( b ) performed 96 h after transduction, confirmed the efficacy of HDAC6 silencing by both constructs. HDAC6 mRNA levels were normalized setting at one the shRNA sample. c MOLT3 or TALL1 cells expressing shHDAC6 #1 or shHDAC6 #2 showed reduced Notch3 FL protein levels. Western blot analysis was not performed in SUPT11 cells as it is known that these cells do not express detectable Notch3 . d HDAC6 silencing was associated with increased apoptosis of T-ALL cells which express Notch3, but not in SUPT11 cells. Apoptosis was analyzed by caspase 3–7 assay 5 days after transduction of cells with the indicated LV. Relative Luminescence Units (RLU) are reported (* P < 0.05; ** P < 0.01, *** P < 0.001, mean ± SD, three independent experiments)

Article Snippet: MOLT3 and Jurkat cell lines were purchased from ATCC (Manassas, VA, USA); DND 41 and TALL1 cell lines were kindly provided by academic colleagues; SUPT11 cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany); all these cell lines were cultured in complete RPMI medium, as reported elsewhere [ ].

Techniques: Transduction, shRNA, Quantitative RT-PCR, Western Blot, Construct, Expressing

Journal: EMBO Reports

Article Title: Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

doi: 10.15252/embr.202152972

Figure Lengend Snippet: A–D Acanthamoeba castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of P flaA (P tac ‐ mCherry ‐P flaA ‐ gfp ) and mCherry constitutively, (B) GFP under the control of P ralF (P ralF ‐ gfp ) and stained with DAPI, (C) GFP under the control of P sidC (P sidC ‐ gfp ) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post‐infection and analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP‐positive) emerged at the colony cluster periphery only after bacterial replication had ceased (red fluorescent Timer). E, F Optical section (E) (scale bar: 5 µm) and distribution of the fluorescence signal intensity (F) of A. castellanii infected with mCherry‐producing L. pneumophila JR32 (P tac ‐ mCherry ‐P flaA ‐ gfp ) for 48 h. The plot profile was analyzed for an optical section in the center of an infected host cell and shows the intensity of the green and red channels, respectively (representative of n = 10). The GFP signal mainly localizes to the periphery of the bacterial cluster. G Transmission electron micrographs of A. castellanii infected with L. pneumophila . Acanthamoeba castellanii was infected (MOI 5, 42 h) with L. pneumophila wild‐type strain JR32 and subjected to high‐pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post‐infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm and 0.5 µm (insets).

Article Snippet: Acanthamoeba castellanii amoebae (ATCC 30234, laboratory collection) were grown in proteose, yeast extract, glucose (PYG) medium at 23°C.

Techniques: Infection, Control, Staining, Confocal Microscopy, Bacteria, Fluorescence, Transmission Assay, Electron Microscopy, Membrane, Clinical Proteomics

Journal: EMBO Reports

Article Title: Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

doi: 10.15252/embr.202152972

Figure Lengend Snippet: A Legionella pneumophila subpopulation proteomics. Acanthamoeba castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (P tac ‐ mCherry ‐P flaA ‐ gfp ). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry‐positive/GFP‐negative and mCherry‐positive/GFP‐positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP‐producing subpopulation have a positive log 2 ratio (and a low q ‐value). B, C The ratio (GFP/mCherry) of selected proteins is indicated for (B) proteins relevant for flagellum formation and (C) cell division proteins and a T4SS substrate (LegC8). The ratio of the mean intensities from three independent proteomics experiments is shown. D, E Expression of select L. pneumophila genes in amoebae. Acanthamoeba castellanii was infected (MOI 5; 6 and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) P fleQ ‐ mCherry ‐P flaA ‐ gfp or (E) P legC8 ‐ mCherr y‐P flaA ‐ gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).

Article Snippet: Acanthamoeba castellanii amoebae (ATCC 30234, laboratory collection) were grown in proteose, yeast extract, glucose (PYG) medium at 23°C.

Techniques: Infection, Lysis, Bacteria, Flow Cytometry, Quantitative Proteomics, Expressing, Construct, Confocal Microscopy

Journal: EMBO Reports

Article Title: Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

doi: 10.15252/embr.202152972

Figure Lengend Snippet: A Time lapse microscopy of A. castellanii infected with P flaA ‐GFP‐producing L. pneumophila . Acanthamoeba castellanii amoebae were infected (MOI 5) with L. pneumophila JR32 (P tac ‐ mCherry ‐P flaA ‐ gfp ) and embedded in 0.5% agarose/PYG medium. Approximately 46 h post‐infection, GFP‐positive (motile) bacteria emerged in the mCherry‐positive intracellular cluster, which within 1.5 h covered the bacterial cluster and spread from the bursting amoeba. At 55 h p.i., primarily mCherry‐positive/GFP‐negative (non‐motile) L. pneumophila remained clustered. Scale bar: 10 µm. B Legionella pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. Acanthamoeba castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆ flaA, or ∆ lqsA harboring P tac ‐ mCherry ‐P flaA ‐ gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 ( n = 25), ∆ flaA ( n = 13), or ∆ lqsA ( n = 19). Scale bars: 20 µm. C, D Histograms of the (C) number and (D) spreading distance of L. pneumophila released from infected amoebae. Spreading was quantified using the Imaris software (“surface” tool). One mask covered the heavily infected amoebae (center: x , y , z = 0), and another mask covered each single bacterium outside the infected host cell. The total number of released bacteria were for JR32 ( n = 1,240), ∆ flaA ( n = 33), and ∆ lqsA ( n = 214). The distance of each bacterium to the center was quantified by vector calculations. The red line marks the approximate cell diameter (5 µm). Data shown are means and SEM of four infected amoebae from one biological replicate (two‐tailed Student's t ‐test; * P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Acanthamoeba castellanii amoebae (ATCC 30234, laboratory collection) were grown in proteose, yeast extract, glucose (PYG) medium at 23°C.

Techniques: Time-lapse Microscopy, Infection, Bacteria, Fluorescence, Software, Plasmid Preparation, Two Tailed Test

Journal: EMBO Reports

Article Title: Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

doi: 10.15252/embr.202152972

Figure Lengend Snippet: A, B Role of the Lqs system for P flaA expression in intracellular L. pneumophila . Acanthamoeba castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS ‐∆ lqsT harboring P tac ‐ mCherry ‐P flaA ‐ gfp , and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed, and analyzed by flow cytometry. C–F Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila . (C, D) Acanthamoeba castellanii or (E, F) Dictyostelium discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS ‐∆ lqsT harboring P tac ‐ mCherry ‐P flaA ‐ gfp , fixed, and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; Δ lqsA : n = 834; Δ lqsR : n = 572; Δ lqsS : n = 729; Δ lqsT : n = 1,469; Δ lqsS ‐ ΔlqsT : n = 693; (F): JR32: n = 136; Δ lqsA : n = 201; Δ lqsR : n = 303; Δ lqsS : n = 183; Δ lqsT : n = 534; Δ lqsS ‐Δ lqsT : n = 180. G, H Dictyostelium discoideum producing the LCV/PtdIns(4) P probe P4C‐GFP and cytosolic mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS ‐∆ lqsT, or ∆ flaA harboring P tac ‐ mCerulean . Representative images of (G) D. discoideum infected with L. pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%), and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; Δ lqsA : n = 468; Δ lqsR : n = 344; Δ lqsS : n = 325; Δ lqsT : n = 287; Δ lqsS ‐ ΔlqsT : n = 289; Δ flaA : n = 248. Data information: Data shown (B, D, F, H) are means and SEM from three biological replicates, (B) done in technical triplicate each (two‐tailed Student's t ‐test; * P < 0.05, ** P < 0.01, *** P < 0.001, or not significant, n.s.).

Article Snippet: Acanthamoeba castellanii amoebae (ATCC 30234, laboratory collection) were grown in proteose, yeast extract, glucose (PYG) medium at 23°C.

Techniques: Expressing, Infection, Confocal Microscopy, Flow Cytometry, Ubiquitin Proteomics, Bacteria, Membrane, Two Tailed Test

Journal: EMBO Reports

Article Title: Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

doi: 10.15252/embr.202152972

Figure Lengend Snippet: A, B Legionella pneumophila JR32, ∆ lqsA , or ∆ lqsR expressing P lqsA ‐ gfp (pCM005) were diluted in AYE broth and incubated for 17 h (replicative phase), 21 h (switch to stationary phase), or 25 h (stationary phase) at 37°C. At the time points indicated, bacteria were collected and fixed, and GFP production was analyzed by (A) microscopy (scale bars: 25 µm) and (B) flow cytometry/FlowJo software. C, D Acanthamoeba castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32, ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS ‐∆ lqsT expressing P lqsA ‐ gfp (pCM005), stained with DAPI, and (C) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 7.5 µm), or (D) lysed, fixed, and analyzed by flow cytometry/FlowJo software. E Acanthamoeba castellanii was infected (MOI 5) for the time indicated with L. pneumophila JR32, ∆ lqsA , or ∆ lqsR expressing P lqsA ‐ gfp (pCM005), stained with DAPI, fixed, and analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). After 6 h of infection, some bacteria are still LqsA‐positive, but then up to 48 h, barely any P lqsA expression is observed. Data information: Data shown are combined means and SEM from (B) three biological replicates, each done in technical triplicate and (D) a technical triplicate (two‐tailed Student's t ‐test; ** P < 0.01, and not significant, n.s.).

Article Snippet: Acanthamoeba castellanii amoebae (ATCC 30234, laboratory collection) were grown in proteose, yeast extract, glucose (PYG) medium at 23°C.

Techniques: Expressing, Incubation, Bacteria, Microscopy, Flow Cytometry, Software, Infection, Staining, Confocal Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a Seahorse measurement of basal oxygen consumption rate (OCR) in MOLM14 ( n = 11) and U937 ( n = 7) AML cell lines, in primary AML patient cells ( n = 4) and in primary normal hematopoietic cells (PBMC n = 6; CD34 + n = 3) treated or not with Etx (3 µM, 15 min; one-sample t -test). b MOLM14 cells were treated with 3-methyladenine (3-MA, 5 mM, 24 h), fixed and stained for Bodipy 493/503 and DAPI. Representative confocal pictures from three independent experiments are shown. Scale bar: 10 µm. c , d MOLM14 ( n = 3) and U937 ( n = 4) AML cell lines, primary AML patient cells ( n = 10) and primary normal hematopoietic cells (PBMC n = 7) were treated or not with 3-MA (5 mM, 24 h), fixed, and stained for Bodipy 493/503 and DAPI. Histograms show the number ( c ) or the area ( d ) of Bodipy 493/503 dots per cell (one-sample t -test). e , g MOLM14 cells were either transduced with a shRNA directed against ATG12 or transfected with a siRNA targeting Beclin1. Cells were then stained for Bodipy 493/503 and DAPI. Representative confocal pictures from three independent experiments are shown ( e ). Scale bar: 10 µm. Graphs represent the number ( f ) or the area ( g ) of Bodipy 493/503 dots per cell, ( n = 3, unpaired t -test). h MOLM14 ( n = 3) and U937 ( n = 5) cells were treated with 3-MA (5 mM, 24 h), and processed for triglycerides content analysis. Graph represents the ratio of triglycerides on total neutral lipids (unpaired t -test). i MOLM14 transduced with Ctrl or ATG12 shRNAs were examined for their rates of β-oxidation, ( n = 3, one-sample t -test). j , k Seahorse measurement of basal OCR in MOLM14 ( n = 3) and U937 ( n = 6) cells treated or not with 3-MA for 24 h ( j ), or MOLM14 cells transfected with siRNA control or targeting Beclin1 ( n = 4) ( k ) (unpaired t -test). Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Staining, Transduction, shRNA, Transfection, Control

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a Volcano plots displaying fold change versus adjusted p -values of MOLM14 (left) and U937 (right) cells treated with 10 mM of metformin (Met) for 24 h ( p- value < 0.05, absolute log2 fold change > 0.5, unpaired t -test). b Venn diagram representing overlap between MOLM14 and U937 downregulated (left) and upregulated (right) genes. Enrichment analysis of Gene Ontology (GO) classification for common downregulated (left) and upregulated (right) genes by Genomatics software analysis (Fisher’s exact test). c MOLM14 cells treated with Met (10 mM) for 24 h were fixed and processed for transmission electron microscopy analysis. Representative electron microscopy pictures from two independent experiments are shown. Arrows indicate lipid droplets. Scale bar: 2 µm. d MOLM14 cells were treated with Met or with antimycin A (AA) and processed for triglycerides content analysis, ( n = 3, unpaired t -test). e MOLM14 cells were treated with Met or with AA, fixed and stained for Bodipy 493/503 and DAPI. Representative confocal pictures from three independent experiments are shown. Scale bar: 10 µm. f , g MOLM14 ( n = 3) and U937 ( n = 3) AML cell lines, primary AML patient cells ( n = 14) and primary normal hematopoietic cells (PBMC n = 9; CD34 + n = 4) were treated with Met or with AA for 48 h, fixed, and stained for Bodipy 493/503 and DAPI. Histograms show the number ( f ) or the area ( g ) of Bodipy 493/503 dots per cell (unpaired t -test or paired t -test for patients’ samples). Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Software, Transmission Assay, Electron Microscopy, Staining

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a , b Western blots of LC3B and actin from at least three independent experiments of MOLM14 cells treated with metformin (Met) ( a ) or with antimycin A (AA) ( b ) ± chloroquine (chloro) for the indicated times are shown. c , d LC3B-II/actin ratios identified by densitometries from Western blots shown in a , b in MOLM14 cells treated with Met ( c ) or with AA ( d ) in presence of chloro. Data are means ± s.e.m, (at least n = 3, unpaired t -test). e Western blots of LC3B and actin from primary AML ( n = 8) or normal ( n = 5) cells treated with Met or with AA ± chloro. f Primary AML patient cells ( n = 8 with Met, n = 8 with AA) and primary normal hematopoietic cells (PBMC) were treated with Met ( n = 5) or AA ( n = 5) for 48 h in presence of chloro followed by immunoblotting for LC3B and actin. Histograms represent the LC3B/actin ratios obtained by densitometric analysis of Western blots (paired t -test). g , h Representative confocal pictures from three independent experiments of MOLM14 cells treated with Met for 48 h ± chloro, fixed and stained for LC3B and DAPI. Scale bar: 10 µm. g Histograms represent the number of LC3B puncta per cell ( h ), ( n = 3, unpaired t -test). i Primary AML patient cells ( n = 8) and primary normal hematopoietic cells (PBMC n = 8, CD34 + n = 4) were treated or not with Met for 48 h ± chloro, fixed and stained for LC3B and DAPI. Histograms represent the number of LC3B puncta per cell (paired t -test). Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Western Blot, Staining

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a MOLM14 cells treated with metformin (Met, 10 mM) for 24 h were fixed and processed for electron microscopy analysis. Representative electron microscopy pictures from two independent experiments are shown. Arrows indicate MERCs. Scale bar: 1 µm. b Histograms represent the % of mitochondria that are in contact with endoplasmic reticulum per cell treated or not with Met from pictures displayed in a . Data are means ± s.e.m with each dot corresponding to one cell. c Protein components of subcellular fractions (left panel) from two independent experiments prepared from MOLM14 cells revealed by immunoblot analysis (right panel). TL: total lysate, Cy: cytosol, Mt: pure mitochondrial fraction, MERCs: mitochondria–ER contact site fraction. d Representative orthogonal confocal projections of Z sections of PLA (red signal) between VDAC1 and IP3R1 from MOLM14 cells treated or not with Met or antimycin A (AA) for 48 h (at least n = 3). Scale bar: 10 µm e Quantitative analysis of PLA signal between VDAC1 and IP3R1 from MOLM14 cells treated or not with Met or AA for 6 h, 24 h or 48 h ( n = 4, one-sample t -test). Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Electron Microscopy, Western Blot

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a MOLM14 cells transfected with siRNA control (Ctrl) or mitofusin2 (Mtfn2) were processed for electron microscopy analysis. Electron microscopy pictures from one experiment are shown. Scale bar: 1 µm. b Histograms represent the % of mitochondria in contact with ER. Each dot corresponding to one cell. c Images of proximity ligation assay between VDAC1 and IP3R1 from MOLM14 transfected with Ctrl or Mtfn2 siRNAs ( n = 3). Scale bar: 10 µm. Numbers represent the % of dots in cells transfected with siRNA Mtfn2 compared with cells transfected with siRNA Ctrl. d , e MOLM14 cells were transduced with Ctrl or VDAC1 shRNAs ( n = 4) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6), and stained for LC3B and DAPI. Confocal sections (at least four independent experiments) are shown. Scale bar: 10 µm ( d ). Histograms represent the number of LC3B puncta per cell ( e ) (unpaired t -test). f , g MOLM14 cells transduced with Ctrl or VDAC1 shRNAs ( n = 3) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6) were stained for Bodipy and DAPI. Scale bar: 10 µm, ( f ). Histograms show the number or the area of Bodipy dots per cell ( g ) (unpaired t -test). h Measurement of basal oxygen consumption rate (OCR) in MOLM14 cells transduced with Ctrl or VDAC1 shRNAs ( n = 6) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6, unpaired t -test). i , j MOLM14 cells transfected with Ctrl or Beclin1 (Bec) or Mtfn2 siRNAs and incubated with RC 12 , were stained for TOMM20 and confocal Z- stacks were acquired (three independent experiments). Scale bar: 10 µm ( i ). Fraction of RC 12 overlapping TOMM20 staining ( j ). siCtrl ( n = 17), siBec ( n = 18), and siMtfn2 ( n = 13, unpaired t -test). k Measurement of OCR in MOLM14 cells transfected with Ctrl ( n = 8), Bec ( n = 7), or Mtfn2 siRNAs ( n = 8) ± Oleate-BSA. l – o MOLM14 cells were transduced with the mitochondria–ER organelle linker (OMM-ER) and its control (OMM), treated ± metformin, and subjected to Western blot analysis for actin and LC3B ( l , m ) or stained for Bodipy and DAPI ( n , o ) (three independent experiments). Scale bar: 10 µm. Histograms represent the number of LC3B puncta ( m ) or the area of Bodipy dots per cell ( o ) unpaired t -test. Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Transfection, Control, Electron Microscopy, Proximity Ligation Assay, Transduction, Staining, Incubation, Western Blot

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a , b NSG mice ( n = 31) were engrafted with MOLM14 cells expressing the Ctrl or VDAC1 shRNAs by intravenous injection. Seventeen days post graft, five mice shCtrl and six mice shVDAC1 were killed, and the number of human cells (hCD45+ and hCD33+) in the bone marrow and spleen was analyzed by flow cytometry. Graphs represent the number of human positive cells for hCD45 and hCD33 within the murine bone marrow and spleen from one experiment ( a ) (unpaired t -test). The remaining mice per group were used for overall survival analysis ( b ). Graph represents the Kaplan–Meier survival curves (Log-rank test). c , d NSG mice ( n = 39) were engrafted with MOLM14 cells and daily treated with vehicle or IACS-010759 by gavage. Seventeen days post graft, nine mice vehicle group and 11 mice IACS-010759-treated group were killed, and the number of human cells (hCD45+ and hCD33+) in the bone marrow and spleen was analyzed by flow cytometry. Graphs represent the number of human positive cells for hCD45 and hCD33 within the murine bone marrow and spleen ( c ) (unpaired t -test). The remaining mice were used for overall survival analysis ( d ). Graph represents the Kaplan–Meier survival curves (Log-rank test). e – g Viable bone marrow AML blasts from mice treated with IACS-010759 were stained for Bodipy 493/503 and DAPI. Scale bar: 10 µm ( e ). Histograms show the number or the area of Bodipy 493/503 dots per cell ( f , g ) (unpaired t -test). h Viable bone marrow AML blasts from mice treated with IACS-010759 (one experiment) were subjected to Western blot analysis for actin and LC3B. Numbers indicate ratios obtained by densitometric analysis. i Purified viable bone marrow AML blasts from mice treated with IACS-010759 (one experiment) were subjected to PLA assay. Numbers represent the number of PLA dots per cell (minimum 130 cells, unpaired t -test). Scale bar: 10 µm ( j ). Schematic diagram depicting the interplay between the autophagy process and the mitochondria in AML cells. Autophagy appears as a major regulator of mitochondria activity with the mitochondria controlling its supply of FFAs by regulating the number of autophagosomes via the formation MERCs. Data are means ± s.e.m.

Article Snippet: The human myeloid leukemia cell lines, MOLM14 and U937 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Leibniz, Germany).

Techniques: Expressing, Injection, Flow Cytometry, Staining, Western Blot, Purification, Activity Assay