Journal: Cell cycle (Georgetown, Tex.)

Article Title: Delphinidin, a dietary anthocyanidin in pigmented fruits and vegetables: a new weapon to blunt prostate cancer growth.

doi: 10.4161/cc.7.21.6969

Figure Lengend Snippet: Figure 2. Effect of delphinidin on apoptotic biomarkers in 22Rν1 cells. 22Rν1 cells were treated with vehicle alone (0.1% DMSO) or specified concentrations of delphinidin in 0.1% DMSO for 48 hours as detailed in Materials and Methods. (A) Protein levels of PARP, cleaved PARP, Bcl2, Bax and active caspase-3 and -9 in 22Rν1 cells as determined by immunoblot analysis. Equal loading of protein was confirmed by stripping and reprobing the blots with β-actin antibody. The data are representative of three independent experiments with similar results. (B) 22Rν1 cells were treated with 10 μM concentration of a general caspase inhibitor Z-VAD- FMK for 4 hours, followed by the treatment with indicated doses of delphinidin for 48 hours. Green fluores- cence of Annexin V staining represents the cells under going apoptosis.

Article Snippet: The general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluromethylketone (Z-VAD-FMK) was obtained from R&D Systems, Inc., (Minneapolis, MN).

Techniques: Western Blot, Stripping Membranes, Concentration Assay, Staining

Journal: Cell Death & Disease

Article Title: ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

doi: 10.1038/s41419-021-03427-y

Figure Lengend Snippet: a Abin-1 +/+ MEFs and Abin-1 −/− MEFs were treated with 10 μg/ml poly(I:C) for 6 h, 7 h, or 24 h in presence or absence of necrostatin-1 (Nec-1s) and then the supernatant was subjected for ToxiLight assay. b Abin-1 +/+ MEFs and Abin-1 −/ − MEFs were treated with 5Z-7-oxozeaenol (5Z-7), 3 μg/ml or 10 μg/ml poly(I:C) (P), 10 ng/ml LPS, 3 μg/ml poly(I:C) + 5Z-7, 10 μg/ml poly(I:C) + 5Z-7, 10 ng/ml LPS + 5Z-7 for 5 h. c Abin-1 +/+ MEFs and Abin-1 −/− MEFs were treated with poly(I:C), poly(I:C) + 5Z-7, poly(I:C) + 5Z-7+zVAD, LPS + 5Z-7, LPS + 5Z-7+zVAD for 5 h in presence or absence of Nec-1s. d , e Tak1 +/+ MEFs and Tak1 − / − MEFs were transfected with negative control (NC) siRNA or Abin-1 siRNA for 36 h followed by stimulation with poly(I:C), poly(I:C) + 5Z-7 (P5) for 6 h in presence or absence of Nec-1s. Tak1 +/+ MEFs ( d ); Tak1 −/− MEFs ( e ). f Abin-1 +/+ MEFs and Abin-1 −/− MEFs were treated with P5 or poly(I:C) + 5Z-7+zVAD (P5Z) for 4 h in presence or absence of Nec-1s, and cells were lysed for western blot analysis for apoptosis markers cleaved caspase-3, cleaved PARP-1, cleaved caspase-8, and cleaved CYLD. g Abin-1 +/+ Ripk1 +/+ , Abin-1 −/− Ripk1 +/+ , Abin-1 −/− Ripk1 +/D138N , and Abin-1 − /− Ripk1 D138N/D138N MEFs were treated with P5, P5Z for 5 h in the presence or absence of Nec-1s. D138N mutation on RIPK1 leads to RIPK1 kinase death. h Abin-1 +/+ and Abin-1 − / − MEFs were treated with P5 for 4.5 h or 7.5 h in the presence or absence of Nec-1s, and then cells were subjected to immunoprecipitation with phospho-RIPK1 S166 antibody followed by western blot analysis of RIPK1. i – k Abin-1 +/+ and Abin-1 −/− MEFs were treated with P5 for 4 h ( i ), 6 h ( j ), or 24 h ( k ) in the presence or absence of Nec-1s, RIPK3 inhibitor GSK-872, or Nec-1s+GSK-872. Supernatants were collected at indicated time points for ToxiLight assay. l , m Abin-1 +/+ and Abin-1 − / − MEFs were transfected with NC siRNA, Ripk3 siRNA, or Mlkl siRNA for 36 h and treated with P5 and P5Z for 5 h ( l ) or 7.5 h ( m ) in the presence or absence of Nec-1s. n Abin-1 +/+ Ripk3 +/+ , Abin-1 − /− Ripk3 +/+ , and Abin-1 −/− Ripk3 − / − MEFs were treated with P5, P5Z for 7.5 h in the presence or absence of Nec-1s. o Abin-1 +/+ MEFs and Abin-1 − / − MEFs were treated with P5 or P5Z for 5 h in the presence or absence of Nec-1s, and then cells were lysed for western blot analysis of p-MLKL, MLKL, cleaved PARP-1, and cleaved caspase-3. p Abin-1 +/+ MEFs and Abin-1 − / − MEFs were treated with P5 for 0–16 h, and cells were lysed for western blot analysis of p-MLKL, MLKL, cleaved PARP-1, and cleaved caspase-3. q Abin-1 −/− MEFs were pre-incubated with 1 μg/ml or 4 μg/ml TNF blocking antibody (TNF Ab) for 45 min, and then cells were incubated with P5 or TNF + 5Z-7 (T5) for 6 h. r Abin-1 +/+ , Abin-1 +/ − , and Abin-1 −/ − MEFs were pre-incubated with XIAP inhibitor SM-164 for 45 min, and then cells were incubated with P5 for 5 h. s Abin-1 +/+ , Abin-1 +/− , and Abin-1 − /− MEFs were treated with poly(I:C) for 0 h and 4 h, and cells were subjected to RNA extraction and qPCR analysis of TLR3. t Abin-1 +/+ and Abin-1 −/ − MEFs were transfected with NC, Tlr3, or Rig-I siRNA for 36 h and then treated with P5 or P5Z for 6 h. Poly(I:C), 10 μg/ml (unless otherwise indicated); 5Z-7, 0.5 μM; Nec-1s, 10 μM; zVAD, 20 μM; and GSK-872, 3 μM. * P < 0.05, ** P < 0.01, or *** P < 0.001.

Article Snippet: Human recombinant TNFα (R&D Systems, Cat#210-TA-005); Recombinant mouse mTNFα (Cell sciences, Cat# CRT192C); Cycloheximide (Sigma, Cat# C-6255); SM-164 (Selleckchem, custom-synthesized); Birinapant (Selleck Chemicals, Cat# S7015); zVAD (Selleck Chemicals, Cat# S8102); Nec-1s (7-Cl-O-Nec-1) (Selleck Chemicals, Cat# S8641); GSK-872 (Sigma, Cat# 5303890001); Necrosulfonamide (Sigma, 480073); LPS (Sigma, Cat# L4391); IDN-6556 (Selleck Chemicals, Cat# S7775); Poly(I:C) (InvivoGen, Cat# tlrl-pic-5); 5Z-7-Oxozeaenol (Sigma, Cat# O9890); 5-fluorouracil (Selleck Chemicals, Cat# S1209); Necrosulfonamide (Selleck Chemicals, Cat# S825); and ELISA Kit for Human TNFα (BioLegend, Cat# 430206).

Techniques: Transfection, Negative Control, Western Blot, Mutagenesis, Immunoprecipitation, Incubation, Blocking Assay, RNA Extraction

Journal: Cell Death & Disease

Article Title: ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

doi: 10.1038/s41419-021-03427-y

Figure Lengend Snippet: a COLO205 cells were transduced with lentivirus-based control shRNA or Abin-1 shRNA and screened with puromycin for 5 days. Then, cells were treated with poly(I:C) + 5Z-7-oxozeaenol (P5) or poly(I:C) + 5Z-7-oxozeaenol+IDN-6556 (P5I) with or without Nec-1s or necrosulfonamide (NSA) for 24 h, and cell deaths were measured by ToxiLight assay. b Time course of P5I-induced cell death. NC shRNA and Abin-1 shRNA COLO205 cells were treated with P5I for 12, 18, or 24 h with or without Nec-1s or NSA. c COLO205 NC shRNA and Abin-1 shRNA cells were treated with P5 for 4 h or 8 h with or without Nec-1s, and then cells were lysed at indicated time points, followed by western blot analysis of ABIN-1 and apoptosis markers cleaved caspase-3 and PARP-1 and necroptosis marker p-MLKL. d COLO205 NC shRNA and Abin-1 shRNA cells were treated with P5I for 4 h or 8 h with or without Nec-1s, and then cells were lysed at indicated time points, followed by western blot analysis of ABIN-1, RIPK1, MLKL, and necroptosis markers p-RIPK1 and p-MLKL. e Comparison of poly(I:C) (P)-, 5Z-7-oxozeaenol -, IDN-6556 (I)-, zVAD (Z)-, poly(I:C) + zVAD (PZ)-, poly(I:C) + IDN-6556 (PI)-, poly(I:C) + 5Z-7+zVAD (P5Z)-, and P5I-induced cell deaths. Drug treatments for 24 h. f COLO205 NC shRNA and Abin-1 shRNA cells were treated with P5I for 0–8 h, and the total RNA was extracted and subjected to the qPCR assay of TLR3 expression. g COLO205 NC shRNA and Abin-1 shRNA cells were treated with P5I for 0–12 h, and cells were lysed and subjected to western blot analysis of ABIN-1 and TLR3. h , i COLO205 NC shRNA and Abin-1 shRNA cells were transfected with NC siRNA or Tlr3 siRNA and incubated for 24 h, followed by P5I treatment for another 24 h in the presence or absence of Nec-1s. Cell deaths were detected by ToxiLight assay ( h ), and knockdown efficiency of TLR3 and ABIN-1 were analyzed by western blot ( i ). Poly(I:C), 10 μg/ml; 5Z-7, 0.5 μM; IDN-6556, 2.5 μM; Nec-1s, 10 μM; zVAD, 20 μM; and NSA, 5 μM. * P < 0.05, ** P < 0.01, or *** P < 0.001.

Article Snippet: Human recombinant TNFα (R&D Systems, Cat#210-TA-005); Recombinant mouse mTNFα (Cell sciences, Cat# CRT192C); Cycloheximide (Sigma, Cat# C-6255); SM-164 (Selleckchem, custom-synthesized); Birinapant (Selleck Chemicals, Cat# S7015); zVAD (Selleck Chemicals, Cat# S8102); Nec-1s (7-Cl-O-Nec-1) (Selleck Chemicals, Cat# S8641); GSK-872 (Sigma, Cat# 5303890001); Necrosulfonamide (Sigma, 480073); LPS (Sigma, Cat# L4391); IDN-6556 (Selleck Chemicals, Cat# S7775); Poly(I:C) (InvivoGen, Cat# tlrl-pic-5); 5Z-7-Oxozeaenol (Sigma, Cat# O9890); 5-fluorouracil (Selleck Chemicals, Cat# S1209); Necrosulfonamide (Selleck Chemicals, Cat# S825); and ELISA Kit for Human TNFα (BioLegend, Cat# 430206).

Techniques: Transduction, Control, shRNA, Western Blot, Marker, Comparison, Expressing, Transfection, Incubation, Knockdown

Journal: Cell Death & Disease

Article Title: ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

doi: 10.1038/s41419-021-03427-y

Figure Lengend Snippet: a – d Four different human CRC cell lines were treated with TNF + birinapant+zVAD (TBZ) or TNFα + birinapant+IDN-6556 (TBI) for 8 h in the presence or absence of Nec-1s. Cells were lysed by RIPA buffer at 8 h and necroptosis markers phospho-RIPK1 (p-RIPK1), RIPK1, phospho-MLKL (p-MLKL), and MLKL were detected by western blot. TNF, 10 ng/ml; birinapant, 100 nM; zVAD, 20 μM; Nec-1s, 10 μM; and IDN-6556, 2.5 μM. Caco-2, human colorectal adenocarcinoma cell ( a ); HCT116, human colorectal carcinoma cell line ( b ); HT-29, human colorectal adenocarcinoma cells ( c ); and COLO205, human colorectal adenocarcinoma cells ( d ). e – g HT-29 cells were transfected with two different Abin-1 siRNAs and incubated for 24 h. Then, cells were re-plated and treated with TNF + birinapant + IDN-6556 (TBI) or TNF + birinapant+zVAD (TBZ) for 8 h in the presence or absence of Nec-1s. Cell deaths were measured by ToxiLight assay. TBI treatments + /− Nec-1s ( e ); TBZ treatments + /− Nec-1s ( f ); and Knockdown efficiency in HT-29 cells ( g ). Proteins were extracted from HT-29 cells 32 h after siRNAs transfection. Each siRNA sample was loaded in duplicates. h – i COLO205 cells were transfected with two different Abin-1 siRNAs and incubated for 24 h. Then, cells were re-plated and treated with TBI or TBZ for 8 h in the presence or absence of Nec-1s. Cell deaths were measured by ToxiLight assay ( h ); knockdown efficiency ( i ). j HT-29 cells were transduced with lentivirus-based control shRNA or Abin-1 shRNA and screened with puromycin for 5 days. Then, cells were treated with TNF + 5-fluorouracil+IDN-6556 (TFI) or TNF + 5-fluorouracil+zVAD (TFZ) for 36 h in the presence or absence of Nec-1s or necrosulfonamide (NSA). k , l HT-29 cells or COLO205 cells were transfected with control siRNA or Abin-1 siRNA and incubated for 24 h. Then, cells were re-plated and treated with TBI for 8 h in the presence or absence of Nec-1s. Cell lyses were subjected to western blot and p-RIPK1, RIPK1, p-MLKL, MLKL, cIAP1, and ABIN-1 were detected. Cell deaths were measured by ToxiLight assay. HT-29 cells TBI treatments + /− Nec-1s (k) ; and COLO205 cells TBI treatments + /− Nec-1s ( l ). TNFα, 10 ng/ml; birinapant, 100 nM; zVAD, 20 μM; Nec-1s, 10 μM; IDN-6556, 2.5 μM; NSA, 5 μM; and 5-FU, 250 μM. * P < 0.05, ** P < 0.01, or *** P < 0.001.

Article Snippet: Human recombinant TNFα (R&D Systems, Cat#210-TA-005); Recombinant mouse mTNFα (Cell sciences, Cat# CRT192C); Cycloheximide (Sigma, Cat# C-6255); SM-164 (Selleckchem, custom-synthesized); Birinapant (Selleck Chemicals, Cat# S7015); zVAD (Selleck Chemicals, Cat# S8102); Nec-1s (7-Cl-O-Nec-1) (Selleck Chemicals, Cat# S8641); GSK-872 (Sigma, Cat# 5303890001); Necrosulfonamide (Sigma, 480073); LPS (Sigma, Cat# L4391); IDN-6556 (Selleck Chemicals, Cat# S7775); Poly(I:C) (InvivoGen, Cat# tlrl-pic-5); 5Z-7-Oxozeaenol (Sigma, Cat# O9890); 5-fluorouracil (Selleck Chemicals, Cat# S1209); Necrosulfonamide (Selleck Chemicals, Cat# S825); and ELISA Kit for Human TNFα (BioLegend, Cat# 430206).

Techniques: Western Blot, Transfection, Incubation, Knockdown, Transduction, Control, shRNA

Journal: Cell Death & Disease

Article Title: ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

doi: 10.1038/s41419-021-03427-y

Figure Lengend Snippet: a, b HT-29 cells were transduced with lentivirus-based control shRNA (NC shRNA) or Abin-1 shRNA and screened with puromycin for 5 days. Then, cells were treated with TNFα + birinapant+IDN-6556 (TBI), birinapant+IDN-6556 (BI), TNFα + birinapant+zVAD (TBZ), or birinapant+zVAD (BZ) for 12 h or 24 h in the presence or absence of Nec-1s or NSA. Drug treatments for 12 h ( a ); Drug treatments for 24 h ( b ); c HT-29 NC shRNA and Abin-1 shRNA cells were treated with BI for 0, 4, 8, 16, 20, or 24 h, and cell deaths were measured by ToxiLight assay. d HT-29 NC shRNA and Abin-1 shRNA cells were treated with BI for 9 h or 11 h in the presence or absence of Nec-1s. Cell lysates were detected with p-RIPK1, RIPK1, p-MLKL, MLKL, and ABIN-1 by western blot. e HT-29 NC shRNA and Abin-1 shRNA cells were treated with 5-fluorouracil+IDN-6556 (FI) or 5-fluorouracil+zVAD (FZ) for 36 h in the presence or absence of Nec-1s or NSA. f , g HT-29 NC shRNA and Abin-1 shRNA cells were treated with birinapant (B), IDN-6556 (I), BI, or BI + Nec-1s for 12 h, and supernatants were collected for ELISA analysis of TNF ( f ), and cells were lysed for western blot analysis of TNF ( g ). h , i HT-29 NC shRNA and Abin-1 shRNA cells were treated with 5-fluorouracil (F), I, FI, or FI + Nec-1s for 36 h, and supernatants were collected for ELISA analysis of TNF ( h ) and cells were lysed for western blot analysis of TNF ( i ). j HT-29 NC shRNA and Abin-1 shRNA cells were treated with TNF for 0, 15, or 30 min and then immunoprecipitated with TNFR1 antibody, followed by western blot analysis for RIPK1 and A20. TNF, 10 ng/ml for cell death and 50 ng/ml for IP; birinapant, 100 nM; 5-FU, 250 μM; zVAD, 20 μM; Nec-1s, 10 μM; IDN-6556, 2.5 μM; and NSA, 5 μM. * P < 0.05, ** P < 0.01, or *** P < 0.001.

Article Snippet: Human recombinant TNFα (R&D Systems, Cat#210-TA-005); Recombinant mouse mTNFα (Cell sciences, Cat# CRT192C); Cycloheximide (Sigma, Cat# C-6255); SM-164 (Selleckchem, custom-synthesized); Birinapant (Selleck Chemicals, Cat# S7015); zVAD (Selleck Chemicals, Cat# S8102); Nec-1s (7-Cl-O-Nec-1) (Selleck Chemicals, Cat# S8641); GSK-872 (Sigma, Cat# 5303890001); Necrosulfonamide (Sigma, 480073); LPS (Sigma, Cat# L4391); IDN-6556 (Selleck Chemicals, Cat# S7775); Poly(I:C) (InvivoGen, Cat# tlrl-pic-5); 5Z-7-Oxozeaenol (Sigma, Cat# O9890); 5-fluorouracil (Selleck Chemicals, Cat# S1209); Necrosulfonamide (Selleck Chemicals, Cat# S825); and ELISA Kit for Human TNFα (BioLegend, Cat# 430206).

Techniques: Transduction, Control, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation