Journal: bioRxiv

Article Title: Pharmacological PP2A reactivation overcomes multikinase inhibitor tolerance across brain tumor cell models

doi: 10.1101/2022.05.31.494146

Figure Lengend Snippet: A) Schematic illustrating PP2A reactivation predisposed to MKI-induced SL in GB. B) Representative images of colony formation assay in T98G cells under PME-1 deletion (siRNA or CRISPR/Cas9) or NZ-8-061 treatment. Cells were treated with 25 nM UCN-01 (UCN) or left untreated (NT). Immunoblot analysis of PME-1 (lower panel). C) Viability of T98G cells treated with increasing concentration of NZ-8-061 either alone or in combination with 25 nM UCN-01 (UCN) for 72 h. ***P<0.001, Student’s t -test. D) Synergy plot showing the most synergistic area (yellow box) between NZ-8-061 and UCN-01 in T98G cells. The Bliss synergy score is calculated over the whole dose-response matrix. E) Viability of T98G wt and SV40 small t-antigen-expressing (T98G-ST) cells treated with 25 nM UCN-01 (UCN) and 8 µM NZ-8-061, alone or in combination for 72 h. Immunoblot analysis of SV40 small t-antigen (right panel). **P<0.01, ***P<0.001 Student’s t -test. F) Caspase 3/7 activity in T98G cells treated with 8 µM NZ-8-061 alone or in combination with 25 nM UCN-01 (UCN) under caspase inhibitor Z-VAD-FMK (20 µM) for 24 h. ***P<0.001, Student’s t -test. G) Structures of two different classes of SMAPs. H) Viability of T98G cells treated SMAPs, 10 µM DBK-794 and 5 µM DBK-1154, alone or in combination with 25 nM UCN-01 (UCN) for 72 h. **P<0.01, ***P<0.001, Student’s t -test. I) Representative images (left) and quantified data of colony formation assay (right) in U251, U118, A172 and U87MG cells treated with 8 µM NZ-8-061 alone or in combination with UCN-01 (UCN; 200 nM, 25 nM, 50 nM and 500 nM, respectively). n=2 independent experiments, *P<0.05, **P<0.01, ***P<0.001, Student’s t -test.

Article Snippet: After 24 hours, cells were treated with the indicated drugs in combination with pan-caspase inhibitor Z-VAD-FMK (10 mM, Promega).

Techniques: Colony Assay, CRISPR, Western Blot, Concentration Assay, Expressing, Activity Assay

Journal: Cell Death & Disease

Article Title: Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion

doi: 10.1038/cddis.2015.12

Figure Lengend Snippet: Hyperthermia increases TRAIL-induced cell death in a caspase-dependent but mitochondrial-independent manner. ( a ) Schematic representation of the protocol used to stimulate cells with TRAIL. Cells were either stimulated at 37 °C for 6 h, or incubated in the presence (T+HS) or absence (HS) of His-TRAIL for 1 h at 42 °C followed by a 5 h additional incubation at 37 °C. ( b ) Indicated cancer cell lines were stimulated with 500 ng/ml His-TRAIL (TRAIL) or TRAIL and hyperthermia (T+HS) as described above and apoptosis was measured after 6 h after the onset of the stimulation by Hoechst staining. ( c ) MDA-MB-231 cells were pre-incubated or not 30 min with 5 μ M caspase inhibitor z-VAD and stimulated with 50 ng/ml His-TRAIL. Apoptosis was measured after 6 h by Hoechst staining. ( d) Five hundred MDA-MB-231 cells, plated overnight in a 6-well plate, were pre-incubated or not for 30 min with 20 μ M z-VAD prior stimulation or not with 500 ng/ml His-TRAIL at 37 °C or in hyperthermic condition (HS) for 1 h and allowed to recover for a week at 37 °C before staining with methylene blue. ( e ) MDA-MB-231 cells were stimulated or not with 500 ng/ml His-TRAIL as indicated for 2, 4 or 6 h and cytosolic or membrane fractions were isolated (see methods). Expression levels of indicated proteins were detected by immunoblotting. One representative blot is shown ( n =3). ( f ) MDA-MB-231 cells were stimulated as described above with 50 ng/ml His-TRAIL and caspase activities were measured by luminometry 1–8 h after stimulation using caspase-3/7 (DEVD), caspase-8/10 (IETD) or caspase-9 (LEHD) luminogenic substrates. ( g ) MDA-MB-231 cells were transfected with non-targeting (si-Nt), caspase-9 (si-C9) or BID (si-BID) targeting siRNAs. Seventy-two hours after transfection, cells were stimulated or not with 100 ng/ml His-TRAIL and apoptosis was analyzed after 6 h by Annexin V staining and flow cytometry. Caspase-9 and BID expression levels are shown on the right. ( h ) MDA-MB-231 cells, transfected with an empty retroviral vector (Mock) or a vector encoding Bcl-xL, were stimulated as above and apoptosis induced by the indicated stimuli was analyzed by Hoechst. Bcl-xL expression is shown on the right. ( i ) MDA-MB-231 cells were transfected with a non-targeting (si-Nt) or caspase-8 (si-C8) targeting siRNA, stimulated or not 72 h after transfection with 100 ng/ml His-TRAIL and apoptosis was analyzed as above. Caspase-8 expression levels were controlled by immunoblotting. ( b , c , d , g , h and i ) Error bars represent S.D. from at least three independent experiments

Article Snippet: The pan-caspase inhibitor z-VAD-fmk and cycloheximide (Cat# ALX-380-269) were from Enzo Life Science (Villeurbanne, France).

Techniques: Incubation, Staining, Isolation, Expressing, Western Blot, Transfection, Flow Cytometry, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion

doi: 10.1038/cddis.2015.12

Figure Lengend Snippet: Hyperthermia prevents FLIP recruitment to the TRAIL DISC. ( a ) MDA-MB-231 cells, stimulated with 500 ng/ml His-TRAIL at 42 °C and/or at 37 °C for the indicated period of time, were lysed and the TRAIL DISC was immunoprecipitated using an anti-histidine antibody (see methods). DISC components associated with TRAIL were analyzed by immunoblotting as indicated. ( b ) MDA-MB-231 cells were stimulated as above and the initiator caspase-8 was immunoprecipitated. TRAIL DISC components were analyzed by immunoblotting. Alternatively, an isotype control antibody (CTL-Ig) was used for specificity. NT stands for non-treated cell extracts. ( a , b ) One representative blot is shown ( n =3). Black arrows show uncleaved proteins, white arrows indicate cleaved proteins. Grey arrow shows the short c-FLIP isoform

Article Snippet: The pan-caspase inhibitor z-VAD-fmk and cycloheximide (Cat# ALX-380-269) were from Enzo Life Science (Villeurbanne, France).

Techniques: Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion

doi: 10.1038/cddis.2015.12

Figure Lengend Snippet: Hyperthermia-mediated cytosolic c-FLIP depletion is sufficient to sensitize tumor cells to TRAIL apoptosis. ( a ) MDA-MB-231 cells were treated with 500 ng/ml His-TRAIL or not (NT) for the indicated times and temperatures, and c-FLIP expression was analyzed from whole-cell lysate (WCL) by immunoblotting together with caspase-8, -10 and FADD. HSC70 and HSP27 were used as loading and heat shock controls, respectively. Black arrows show uncleaved proteins, white arrows indicate cleaved proteins. Grey arrow shows the short c-FLIP isoform. ( b ) MDA-MB-231 cells were stimulated as above and lysed in NP40 1% lysis buffer. After centrifugation, the supernatants (soluble) and the pellets resuspended in a buffer containing 1% SDS (insoluble), were loaded and indicated proteins were analyzed by immunoblotting. ( c , d ) MDA-MB-231 cells were transfected with non-targeting (si-Nt) or FLIP (si-FLIP) targeting siRNAs and stimulated 72 h after transfection with ( c ) 50 ng/ml or ( d ) increasing concentrations of His-TRAIL and apoptosis (Annexin V) was measured by flow cytometry. FLIP expression levels were controlled by immunoblotting. ( e ) MDA-MB-231 cells stably expressing c-FLIP L or c-FLIP S or infected with an empty vector (Mock) were stimulated with 50 ng/ml His-TRAIL as above and apoptosis (Annexin V) was measured by flow cytometry. FLIP expression levels were controlled by immunoblotting. ( f ) Stably expressing c-FLIP L or Mock-infected MDA-MB-231 cells were stimulated or not (NT) as indicated with 500 ng/ml His-TRAIL for 15–120 min and expression of c-FLIP L was analyzed by immunoblotting in whole-cell lysates (WCL), as well as cytosolic and insoluble NP40 detergent fractions, as described above. ( g ) MDA-MB-231 stably expressing c-FLIP L , c-FLIP S or mock-infected (Mock) cells were stimulated with 500 ng/ml His-TRAIL for 30 min at 37 °C or at 42 °C (T+HS) and caspase-8 was immunoprecipitated for analysis of TRAIL DISC composition by immunoblotting. ( a , b , f , g ) One representative blot is shown ( n =3)

Article Snippet: The pan-caspase inhibitor z-VAD-fmk and cycloheximide (Cat# ALX-380-269) were from Enzo Life Science (Villeurbanne, France).

Techniques: Expressing, Western Blot, Lysis, Centrifugation, Transfection, Flow Cytometry, Stable Transfection, Infection, Plasmid Preparation, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion

doi: 10.1038/cddis.2015.12

Figure Lengend Snippet: Hyperthermia-induced c-FLIP L/S aggregation provokes c-FLIP depletion from the cytosol, thereby preventing its recruitment to the TRAIL DISC and enhancing apoptosis induced by TRAIL. ( a ) The lung carcinoma A549, the liver adenocarcinoma SK-HEP1, and the two colon carcinoma SW480 and HCT116 cell lines were incubated or not (NT) at 42 °C for 15 or 60 min and c-FLIP L expression was analyzed in NP40 soluble and insoluble fractions by immunoblotting. ( b ) Schematic representation of the cell fractionation experiment performed in ( c ). MDA-MB-231 cells were lysed in NP40. Extracts were then either incubated on ice (0 °C) or at 37 °C for 60 min, or incubated for 15 or 60 min at 42 °C and soluble and insoluble fractions were separated by centrifugation to analyze FLIP expression levels by immunoblotting. ( d ) MDA-MB-231 cells were lysed in NP40 and incubated or not at 42 °C for the indicated times in the presence or absence of increasing amounts of glycerol (expressed here as % v/v). Expression levels of c-FLIP L , caspase-8 and caspase-10 were analyzed by immunoblotting. Actin was used as a loading control. ( e and f ) MDA-MB-231 or HCT116 cells were pre-incubated for 60 min with increasing amounts of glycerol and incubated or not at 42°C for 30 minutes. Cells were then lysed in NP40 and cell extracts and centrifuged to collect the soluble and the insoluble NP40 fractions. c-FLIP and caspase-8 content was analyzed by immunoblotting. ( g ) MDA-MB-231 cells were pre-incubated as above in the presence of 5% glycerol and stimulated or not 6 h in the presence of 50 ng/ml His-TRAIL at 37 °C or 42 °C (HS, see ). Apoptosis (Annexin V) was measured by flow cytometry. ( h ) MDA-MB-231 cells were pre-incubated or not for 60 min with glycerol (5% v/v) and stimulated with 500ng/ml His-TRAIL at 37°C or at 42°C (HS). TRAIL DISC was analyzed 2 h after the onset of the stimulation by immunoblotting after immunoprecipitation of the caspase-8. Black arrows show uncleaved proteins, white arrows indicate cleaved proteins. ( a , c , d , e , f , h ) One representative blot is shown ( n =3)

Article Snippet: The pan-caspase inhibitor z-VAD-fmk and cycloheximide (Cat# ALX-380-269) were from Enzo Life Science (Villeurbanne, France).

Techniques: Incubation, Expressing, Western Blot, Cell Fractionation, Centrifugation, Flow Cytometry, Immunoprecipitation