Journal: The FEBS journal

Article Title: Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway.

doi: 10.1111/j.1742-4658.2010.07959.x

Figure Lengend Snippet: Fig. 1. DAPK protein level inversely correlates with TSC2 expression. (A) A549 cells were transfected with increasing amounts of FLAG– TSC2. Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2. (B) Quantification of DAPK protein levels from (A). Results are reported as the mean ± SD (**P < 0.01, n = 3). (C) HEK293 cells were transfected with either TSC2 siRNA or nonspecific control siRNA, as indicated, for 48 h. Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous TSC2, DAPK, S6K P-T389, S6K and actin. (D) Cell lysates were prepared from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs and immunoblotted with antibodies to detect endogenous TSC2, DAPK, S6K P-T389, S6K and actin. (E) TSC2 () ⁄ )) MEFs were transfected with FLAG–TSC2. Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2. (F) Cell lysates were prepared from TSC2 (+ ⁄ +) and TSC2 () ⁄ )) MEFs an assessed for DAPK activity by immunoblotting with antibodies to detect P-Ser308 DAPK, DAPK and actin. (G) A549 cells were transfected with vector control or FLAG–TSC2 and the mRNA levels of DAPK determined by real-time PCR (NS, not significant, n = 3). (H) A549 cells were trans- fected with vector control or FLAG–TSC2. Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK and actin or FLAG antibodies to detect TSC2.

Article Snippet: For immunoprecipitation of endogenous TSC1 from cells, the same procedure was followed except TSC1 antibodies were bound to protein G beads. siRNA For the knockdown of gene expression cells were transfected with TSC2 siRNA (Cell Signaling Technology), ATG7 siRNA (Cell Signaling Technology), Rheb siRNA (Dharmacon, Lafayette, CO, USA), Raptor siRNA (Dharmacon) or a non-specific siRNA as control (Dharmacon).

Techniques: Expressing, Transfection, Control, Activity Assay, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: The FEBS journal

Article Title: Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway.

doi: 10.1111/j.1742-4658.2010.07959.x

Figure Lengend Snippet: Fig. 2. DAPK protein level is not affected by mTORC1 activity. (A) HEK293 cells were treated with 100 nM rapamycin for 6 h. Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6K P-T389, S6K, S6 P-S235 ⁄ 236, S6 and actin. (B) TSC2 () ⁄ )) MEFs were treated with 100 nM rapamycin for the indicated time. Cell lysates were prepared and immunoblotted with antibod- ies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin. (C) HEK293 cells were transfected with control vector or FLAG–Rheb. Cells were serum-starved and treated with 100 nM rapamycin for 2 h where indicated. Cell lysates were prepared and immunoblotted with anti- bodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or FLAG antibodies to detect Rheb. (D) TSC2 () ⁄ )) MEFs were transfect- ed with either Rheb siRNA or nonspecific control siRNA, as indicated, for 48 h. Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous Rheb, DAPK, S6 P-S235 ⁄ 236, S6 and actin. (E) HEK293 cells were transfected with HA–Raptor or HA–Raptor mutant 4. Cells were serum-starved and treated with 100 nM rapamycin for 2 h where indicated. Cell lysates were prepared and immunoblotted with antibodies to detect endogenous DAPK, S6 P-S235 ⁄ 236, S6 and actin or HA antibodies to detect Raptor. (F) TSC2 () ⁄ )) MEFs were transfected with either Raptor siRNA or nonspecific control siRNA, as indicated, for 48 h. Following transfection, cell lysates were prepared and immunoblotted with antibodies to detect endogenous Raptor, DAPK, S6 P-S235 ⁄ 236, S6 and actin.

Article Snippet: For immunoprecipitation of endogenous TSC1 from cells, the same procedure was followed except TSC1 antibodies were bound to protein G beads. siRNA For the knockdown of gene expression cells were transfected with TSC2 siRNA (Cell Signaling Technology), ATG7 siRNA (Cell Signaling Technology), Rheb siRNA (Dharmacon, Lafayette, CO, USA), Raptor siRNA (Dharmacon) or a non-specific siRNA as control (Dharmacon).

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Mutagenesis

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Western blot analyses of ATG7 in different tissue homogenates from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice. b Western blot analyses of ATG7, P62, and LC3-I/II protein expression in VSMCs isolated from the aorta of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice. α-SMA was used as a loading control and is a classical marker for VSMCs. Bands are shown in duplicate for two different primary VSMC cultures. c Representative images of consecutive aortic sinus sections stained with oil red after 10 weeks of high-fat diet (HFD) in Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice. The graph represents the % of plaque area and the data are the mean ± SEM. ** P < 0.01; Student’s t -test, n = 10 mice/group. Scale bar, 100 µm. d Representative images of en face staining of the whole aorta from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 18 weeks of HFD. The graph represents the % of plaque area and the data are the mean ± SEM. * P < 0.05; Student’s t -test, n = 3 mice/group. e Representative images of consecutive aortic sinus sections stained with Masson’s trichrome to assess the total collagen content in Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. The graph represents the % of total collagen content and the data are the mean ± SEM. ns, nonsignificant; Student’s t- test, n = 10 mice/group. Scale bar, 100 µm. f Representative images of consecutive aortic sinus sections stained with Picrosirius red and analyzed using polarized light microscopy to distinguish between type 1 (red) and 3 (green) collagen after 10 weeks of HFD. The graph represents the % of type 3 collagen content and the data are the mean ± SEM. *** P < 0.001; Student’s t -test, n = 8 mice/group. Scale bar, 100 µm

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Western Blot, Expressing, Isolation, Marker, Staining, Light Microscopy

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Representative images of consecutive aortic sinus sections immunostained with MOMA 2 antibody to quantify the content of macrophages/monocytes in the plaque area of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of high-fat diet (HFD). DAPI (blue, nucleus). The graph represents the % of MOMA 2 staining and the data are the mean ± SEM, ** P < 0.01; Student’s t -test, n = 8 mice/group. Scale bar, 200 µm. b Representative images of apoptotic cell detection by TUNEL staining in aortic sinus sections of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. TUNEL (green), DAPI (blue, nucleus). The graph represents the number of TUNEL-positive cells measured in the plaque area and the data are the mean ± SEM. ** P < 0.01; Student’s t -test, n = 8 mice/group. Scale bar, 50 µm. c Representative images of consecutive aortic sinus sections immunostained with cleaved-caspase 3 (green) and α-SMA (red) antibodies to detect apoptotic VSMCs of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. DAPI (blue, nucleus). The graph represents the % of cleaved-caspase 3-positive VSMCs measured in the plaque area and the data are the mean ± SEM. ** P < 0.01; Student’s t -test, n = 8 mice/group. Scale bar, 50 µm

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Staining, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Representative images of consecutive aortic sinus sections stained with MitoSOX (red) and DAPI (blue, nucleus) of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of high-fat diet (HFD). Scale bar, 20 µm. b The graph represents the % of MitoSox staining in the plaque area per section and the data are the mean ± SEM. *** P < 0.001; Student’s t -test, n = 5 mice/group. c Measurement of mitochondrial ROS production in VSMCs isolated from the aorta of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. The graph represents the quantification of MitoSOX Red fluorescence at baseline or after antimycin A (AA, 10 µM) stimulation. Data are the median with interquartile range of four independent experiments from different primary VSMC cultures per group. ** P < 0.01; ## P < 0.01; # P < 0.05; ns, nonsignificant; one-way ANOVA, Kruskal–Wallis non-parametric test. d Measurement of the mitochondrial membrane potential (ΔΨm) with the JC-1 dye in VSMCs isolated from the aorta of Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. The graph represents the quantification of the potential-dependent accumulation of the JC-1 dye in mitochondria at baseline and after CCCP (20 µM) stimulation. Data are the median with interquartile range of four independent experiments from different primary VSMC cultures per group. *** P < 0.001; ## P < 0.01; ns, nonsignificant; one-way ANOVA, Kruskal–Wallis non-parametric test

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Staining, Isolation, Fluorescence

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Representative images of aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of high-fat diet (HFD) and stained with MitoTtracker Deep Red (MitoTR, red), DAPI (blue, nucleus). Scale bar, 5 µm. b The graph represents the calculated average size of mitochondria (µm 2 ) in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Data are the mean ± SEM of 10 cells analyzed from three independent experiments from different primary VSMC cultures per group.*** P < 0.001; Student’s t- test. c Representative images of structured illuminated confocal microscopy (SIM) followed by an image processing algorithm deconvolution and 3D reconstruction. The graph represents the calculated average volume of mitochondria (µm 3 ) stained with MitoTR, in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Data are the mean ± SEM of six cells analyzed from two independent experiments from different primary VSMC cultures per group. *** P < 0.001; Student’s t -test. d Representative images of Ser616 phosphorylated Drp-1 (P-Drp-1, green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. The graph represents the intensity of P-Drp-1 fluorescence. Data are the median with interquartile of six cells analyzed from four independent experiments from different primary VSMC cultures per group. ** P < 0.01; Mann–Whitney non-parametric test. e Seahorse profile for oxygen consumption rate (OCR) in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD with treatment with oligomycin, FCCP, and antimycin A/rotenone. Data are the mean ± SEM of five replicates from three independent experiments from different primary VSMC cultures per group. The graphs represent the basal respiration (last rate measurement before first injection - non mitochondrial respiration rate) and ATP production (last rate measurement before first injection – minimum rate measurement after oligomycin injection) in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD, the data are the mean ± SEM of five replicates from three independent experiments from different primary VSMC cultures per group. * P < 0.05; Student’s t -test

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Isolation, Staining, Confocal Microscopy, Immunostaining, Fluorescence, MANN-WHITNEY, Injection

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Representative images of consecutive aortic sinus sections immunostained with PINK1, Parkin, P62 (red), α-SMA (green) antibodies, and DAPI (blue, nucleus) from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of high-fat diet (HFD). The graph represents the % of PINK1, Parkin, or P62 staining in VSMCs within the plaque area and the data are the mean ± SEM from n = 8 mice/group. *** P < 0.001; Student’s t -test. Scale bar, 20 µm. b Western blot analyses of the expression of PINK1 and Parkin proteins in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD, β-actin was used as the loading control. Bands are shown in duplicate. The graph represents the densitometric analysis of the expression level of PINK1 and Parkin proteins. The data are the mean ± SEM of three independent experiments from different primary VSMC cultures per group. ** P < 0.01; * P < 0.05; Student’s t -test

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Staining, Western Blot, Expressing, Isolation

Journal: Cell Death & Disease

Article Title: Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

doi: 10.1038/s41419-019-1400-0

Figure Lengend Snippet: a Flow cytometry analyses of mitophagy flux in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of high-fat diet (HFD). Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL, left graph) or CCCP (20 µM, right graph) for 8 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). VSMCs were then stained with MitoTR for flow cytometry analysis. The data are expressed as mean ± SEM of five independent experiments (left graph); * P < 0.05; Wilcoxon signed-rank test, ns nonsignificant; and as mean ± SEM of three independent experiments (right graph) * P < 0.05; Mann–Whitney non-parametric test. b Western blot analyses of the expression of TOMM 40 and VDAC1 proteins in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 16 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TOMM 40 and VDAC1 proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. * P < 0.05; *** P < 0.01; one-way ANOVA, Bonferroni’s multiple comparison test. c Western blot analyses of the expression of TFEB and PGC-1-α proteins in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TFEB and PGC-1-α proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. * P < 0.05; *** P < 0.001; Student’s t -test. ( d ) Representative images of TFEB (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. The graph represents the intensity of nuclear TFEB fluorescence and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. * P < 0.05; ### P < 0.001; one-way ANOVA, Kruskal–Wallis non-parametric test. e Representative images of cleaved-caspase 3 (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from Atg7 +/+ Tagln-Cre + , ApoE −/− and Atg7 F/F Tagln-Cre + , ApoE −/− mice after 10 weeks of HFD. Cells were incubated with or without oxidized LDL (200 μg ApoB/mL) for 16 h. Scale bar, 20 µm. The graph represents the % of cleaved-caspase 3-positive cells and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. * P < 0.05; two-way ANOVA, Bonferroni’s multiple comparison test. Scale bar, 20 µm

Article Snippet: After 24 h, DMEM was removed and cells were transduced by self-inactivating lentiviral particles at 20 multiplicity of infection, for the expression of plasmids with a gene coding for green fluorescent protein (GFP), a puromycin resistance gene, and an Atg7-targeted or scramble short hairpin RNA (shRNA) gene (Origene, TL504956V).

Techniques: Flow Cytometry, Isolation, Incubation, Staining, MANN-WHITNEY, Western Blot, Expressing, Immunostaining, Fluorescence

Journal: Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine

Article Title: Effect of zinc intake on hepatic autophagy during acute alcohol intoxication

doi: 10.1007/s10534-018-0077-7

Figure Lengend Snippet: Effect zinc depletion on Atg7, Beclin1 and p4EBP expression in VL17-A cells. (A) Western blot images; (B) Western blot densitometry values of markers of autophagic flux; (C) mRNA expression of MT2A and p62/SQSTM1. Cells were incubated in zinc depleted medium (prepared with FBS treated with Chelex-100), supplemented with 0 or 5 μM zinc for 48h and subsequently exposed to 0 or 100 mM ethanol for 24h. Means without a common letter are significantly different (p < 0.05). * Significantly different (p < 0.05).

Article Snippet: Generation of Atg7 knockout cell line VL17-A cells were transfected with ATG7 CRISPR/Cas9 KO (h) and ATG7 HDR (h) plasmids from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Incubation

Journal: Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine

Article Title: Effect of zinc intake on hepatic autophagy during acute alcohol intoxication

doi: 10.1007/s10534-018-0077-7

Figure Lengend Snippet: Effect of zinc depletion on the protein expression of p62/SQSTM1 in autophagy deficient VL17A (cells lacking Atg7 expression). Cells were treated as described in figure 4 legend. Representative of three independent experiments.

Article Snippet: Generation of Atg7 knockout cell line VL17-A cells were transfected with ATG7 CRISPR/Cas9 KO (h) and ATG7 HDR (h) plasmids from Santa Cruz Biotechnology.

Techniques: Expressing