parp trap assay kit  (BPS Bioscience)

Journal: bioRxiv

Article Title: PARP inhibitors enhance reovirus-mediated cell killing through the death-inducing signaling complex (DISC) with an associated NF-κB-regulated immune response

doi: 10.1101/2023.12.21.572541

Figure Lengend Snippet: A. A375, MeWo and D04 melanoma cells were pre-treated with 0.1 μM talazoparib and thereafter infected at an MOI of 0.1 and 1 of RT3D. Cells were harvested, and the lysates collected at 48 hours post-infection. Poly ADP ribosylation (PAR) was assessed by immunoblotting. B. PAR was also assessed by ELISA. C. CD1 nude mice bearing A375 tumour xenografts were treated with oral administration of vehicle or 0.1 mg/kg talazoparib from Day 1-5. RT3D was injected intratumorally on Day 3 at 1x10 6 pfu, or sham injection. Following the last treatment on day 5, tumours were harvested and assessed for PAR by ELISA. D. RT3D sensitivity was assessed in HeLa PARP-1 paired cells: PARP-1 +/+ (wild type) and PARP-1 -/- (clones G3 and G9), where cytotoxicity was measured by MTT assay 72 hours post-infection. E. Cell viability assays were carried out to assess RT3D plus talazoparib in HeLa PARP-1 paired models (PARP-1 +/+ and PARP-1 -/- ) as shown by crystal violet assays. F. SRB cell viability assay to assess RT3D plus talazoparib in HeLa PARP-1 paired models at 72 hours post-infection. Results are shown from 3 independent experiments. * = p value, **= p value <0.01 and *** = p value <0.001. G. SRB cell viability assay to assess RT3D plus talazoparib in HeLa PARP-1 mutant models (A7 & F7) versus wild type. Results are shown from 3 independent experiments.

Article Snippet: We used the PARP1-Trap Agarose kit [xtak-20] from Chromotek which consists of a PARP1 Nanobody/ VHH, coupled to agarose beads to immunoprecipitate endogenous PARP-1 proteins.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Clone Assay, MTT Assay, Viability Assay, Mutagenesis

Journal: bioRxiv

Article Title: PARP inhibitors enhance reovirus-mediated cell killing through the death-inducing signaling complex (DISC) with an associated NF-κB-regulated immune response

doi: 10.1101/2023.12.21.572541

Figure Lengend Snippet: A. A375 or MeWo cells were treated with RT3D (MOI of 0.1) plus 0.1 μM talazoparib in the presence of either pan-Caspase-, or individual Caspase-inhibitors (all at 1 μM) and thereafter measured for cell survival using MTT. B. A375, MeWo and D04 cells were treated with 0.1 μM talazoparib and thereafter infected with RT3D (MOI of 0.1) for 48 hours. Western analysis was carried out to assess Caspase-8, Caspase-3 and PARP cleavage and modulation of inhibitors of apoptosis, cIAP1 and c-FLIP L . Equal loading of proteins was assessed by probing for α-tubulin. C. Western analysis was carried out in A375 xenograft tumours for PAR, Caspase-8, Caspase-3 and PARP cleavage. Equal loading was measured by probing for α-tubulin. D. Western analysis was carried out in HeLa PARP-1 paired models. PARP-1 +/+ (wild type), and PARP-1 -/- (clones G3 and G9) were infected with RT3D at MOI of 0.1 and 1.0 and immunoblotted for PAR, Casapse-8 and Caspase-3 cleavage. Equal loading was measured by probing for α-tubulin. E. Western analysis was carried out to assess PAR expression and cleavage of Caspase-3 and PARP, following treatment of talazoparib (0.1μM) in HeLa PARP-1 +/ + and PARP-1 -/- (clone G9) cells. RT3D was then infected in the cells at MOI of• (PARP-1 -/- (clone G9) and MOI of 1.0 (PARP-1 +/ + ). Equal loading was measured by probing for α-tubulin. F. Proteomic analysis of A375 cells treated with RT3D (MOI of 0.1) in combination with 0.1 μM talazoparib showed upregulation (red) and down-regulation (green) of the apoptotic death domain pathway. Data are an average of 3 independent experiments. G & H. A375 cells were transfected with scramble control (SC) or siRNA targeting RIPK-1, Caspase-8 or FADD (all at 50 nM) and subsequently treated with 0.1 μM talazoparib and RT3D (MOI of 0.1) for 48 hours and assessed by SRB cell viability assay. I. Western blot analysis was carried out in A375 cells transfected with scramble control (SC) or siRNA targeting Caspase-8, FADD, or RIPKI all at 50 nM and subsequently treated with talazoparib and RT3D at an MOI of 0.1 for 48 hours. Lysates were immunoblotted for PAR, cleaved PARP-1, Caspase-8, FADD or RIPK1 to confirm siRNA target effect. J. A375 cells were pre-treated with 0.1 μM talazoparib and infected with RT3D at an MOI of 0.1 and immunoprecipitation (IP) assay with PAR antibody was carried out. Western analysis was carried out to assess the interaction between PARylated proteins and the DISC components (Caspase-8, FADD, TRADD, RIPK1 and DR5). The input (lysate) was carried out to confirm RT3D induced PARylation (refer to supplementary data). Equal loading of proteins was assessed by probing for α-tubulin. K. Caspase-8 immunoprecipitation was performed in A375 cells. Z-VAD (10 μM) was added in all samples prior to any treatment to prevent destabilisation of complexes with Caspase-8. Cells were then treated with RT3D (MOI of 0.1)/talazoparib (0.1 μM) at 0, 24 and 48 hours. Western analysis was carried out for PAR, RIPK1 and FADD antibodies. The input (lysate) was carried out to confirm expected RT3D induced PARylation and Caspase-8 cleavage (supplementary fig 7).

Article Snippet: We used the PARP1-Trap Agarose kit [xtak-20] from Chromotek which consists of a PARP1 Nanobody/ VHH, coupled to agarose beads to immunoprecipitate endogenous PARP-1 proteins.

Techniques: Infection, Western Blot, Clone Assay, Expressing, Transfection, Viability Assay, Immunoprecipitation