Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Identification of Cholix-interaction proteins on HepG2 cells and immortalised human hepatocytes. (a) After biotinylation of surface proteins, hepatocytes were solubilised with RIPA buffer and immunoprecipitated with heat-inactivated or catalytically inactive mutant Cholix (E581A; C) as described in Section 4. Cholix-binding proteins were detected using streptavidin-HRP. (b) After trypsin hydrolysis, LC-MS/MS analysis of 30-kDa and 33-kDa proteins was performed. The protein had sequences identical to that of proteins, PHB1 and PHB2. (c) Proteins from HepG2 or hepatocytes were immunoprecipitated with Cholix as described above, separated by SDS-PAGE and transferred to PVDF membranes, which were reacted with anti-PHB1 (left) or anti-PHB2 antibodies (right). TCL; total cell lysate. (d) Purified wild-type Cholix (0.5 μg) was incubated for 4 hr with GST-, GST-fused PHB1, or GST-fused PHB2 conjugated beads and then washed with PBS three times. After SDS-PAGE, proteins were transferred to PVDF membranes and visualised by Western blotting using GST-HRP or anti-Cholix antibodies

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Immunoprecipitation, Mutagenesis, Binding Assay, Liquid Chromatography with Mass Spectroscopy, SDS Page, Purification, Incubation, Western Blot

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Effect of Cholix on prohibitin (PHB) expression and localisation in hepatocytes. (a) Hepatocytes were treated for the indicated time points with 100 μg/ml cycloheximide (CHX), 5 μg/ml PEA, mutant Cholix(E581A; mt) or wild-type (wt) Cholix. Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. GAPDH was used as an internal control. Quantification of PHB1 and PHB2 levels in hepatocytes was performed by densitometry (bottom panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments and significance is *p < .05. Experiments were repeated three times with similar results. (b) Hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml), and total RNA was extracted as described in Section 4. PHB1 and PHB2 mRNA were measured by real-time qPCR. Data are shown as mean ± SD of values from two experiments. (c) Hepatocytes (5 × 104 cells/well) in 12-well plates were treated with wt Cholix (5 μg/ml) for the indicated times. Then, cells were fixed and visualised using anti-PHB1 or PHB2 antibodies; mitochondria were seen with MitoTracker as described in Section 4. Nuclei were stained with DAPI. Experiments were repeated three times with similar results. (d) Hepatocytes were treated for 12 hr with 5 μg/ml mt or wt Cholix. Then, cells were lysed with RIPA buffer and then immunoprecipitated with control rabbit IgG (C) or anti-PHB1 monoclonal antibody (P1). The complexes were lysed with 1xSDS sample buffer and analysed by immunoblotting with the indicated antibodies. TCL: total cell lysate. Experiments were repeated three times with similar results. (e) Hepatocytes (5 × 104 cells/well) in 12-well plates were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were fixed, and PHB1 and PHB2 were visualised as described in Section 4. Nuclei were stained with DAPI. Experiments were repeated three times with similar results

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Expressing, Mutagenesis, Western Blot, Control, Incubation, Staining, Immunoprecipitation

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Cholix-induced apoptosis is enhanced in PHB-knockdown hepatocytes. (a) Control (NC) and PHB1 or PHB2 siRNA-transfected hepatocytes were labeled with biotin, lysed with RIPA buffer, and then immunoprecipitated with Cholix or avidin-beads. The complexes were lysed with 1xSDS sample buffer and analysed by immunoblotting with the indicated antibodies. (b) The indicated siRNA-transfected hepatocytes were incubated for 8 hr with mt or wt Cholix (5 μg/ml), lysed with 1xSDS sample buffer and analysed by immunoblotting with antibodies. Quantification of cPARP, PHB1, or PHB2 levels in hepatocytes was performed by densitometry (right panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments, and significance is *p < .05. Experiments were repeated three times with similar results. (c) The indicated siRNA-transfected hepatocytes were incubated for 8 hr with CHX (100 μg/ml) or PEA (5 μg/ml), lysed with 1xSDS sample buffer, and analysed by immunoblotting with antibodies. Quantification of cPARP level in hepatocytes was performed by densitometry (bottom panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments. n.s.: not significant. (d) The indicated siRNA-transfected Hepatocytes (5 × 104 cells/well) in 12-well plates were incubated for 24 hr with wt or mt Cholix (5 μg/ml). Cells were fixed with 4% PFA and then visualised with anti-actin (green) or α-tubulin (red) antibodies. Nuclei were stained with DAPI. The bottom panel of each group represents enlarged version of the white square in middle panel. Experiments were repeated two times with similar results

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Knockdown, Control, Transfection, Labeling, Immunoprecipitation, Avidin-Biotin Assay, Western Blot, Incubation, Staining

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: ROCK1 participates in a Cholix-induced apoptotic pathway. (a) Hepatocytes were pretreated for 30 min with or without 0, 30, 60 μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with anti-cleaved PARP (cPARP) or GAPDH antibodies. A blot representative of three independent experiments is shown. (b) Hepatocytes were pretreated for 30 min with or without 60 μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with anti-cPARP, ROCK1, PHB1, PHB2, or GAPDH antibodies. A blot representative of three separate experiments is shown. (c) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown. (d) Hepatocytes were pretreated for 30 min with or without 60μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were treated with CellROX Deep Red reagent before fixation with 4% PFA and staining with anti-TOM20 (green) or anti-Drp1 (Red) antibodies. Nuclei were stained with DAPI as described in Section 4. Experiments were repeated three times with similar results. (e) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown. (f) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml) in the presence or absence of 20 μM Z-VAD-FMK. Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown.

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Incubation, Western Blot, Transfection, Staining