Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Derivative Assay, Polymer, Plasmid Preparation, Blocking Assay, Membrane, Staining, RNA HS Assay, dsDNA Assay, Protease Inhibitor, Software, Imaging, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1.

doi: 10.1016/j.bbamcr.2012.05.022

Figure Lengend Snippet: Fig. 4. USP2c interacts with and de-ubiquitinates RIP1. (A) USP2c co-immunoprecipitates with endogenous RIP1. MCF7 cells were transfected with plasmids coding WT USP2a-HA. Total cell lysates were left untreated (Ctrl, Input) or immunoprecipitated with an anti-HA antibody and blots probed with the indicated antibodies. (B) USP2c de‐ubiquitinates RIP1 in vivo. 293T cells were transfected with the indicated plasmids coding for WT ubiquitin-HA, K48 ubiquitin-HA or K63 ubiquitin-HA together with USP2c or its inactive mutant. 24 h after transfection cells were treated with MG132 (10 μM, left panels) for 4 h or with TNF (20 ng/ml, right panels) for 15 min and then lysed in a denaturating dissociation buffer. The cell lysates were immunoprecipitated with an anti-RIP1 antibody and blots were probed with the indicated antibodies.

Article Snippet: Mammalian expression vectors coding for human USP2a, human TRAF2 and human RIP1 were from Origene (USA).

Techniques: Transfection, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, Mutagenesis

Journal: Biochimica et biophysica acta

Article Title: De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1.

doi: 10.1016/j.bbamcr.2012.05.022

Figure Lengend Snippet: Fig. 8. The ratio of USP2a and TRAF2 determine the apoptosis sensitivity to TNF. (A) USP2a downregulation reduces and TRAF2 downregulation increases apoptosis by TNF. MCF7 cells were transfected with a siRNA construct against TRAF2 or USP2a, respec- tively or with a scrambled control siRNA (sc) (top panels). 48 h post-transfection, cells were treated overnight with TNF (100 ng/ml). Cells were harvested and stained with PI to quantify cell death (bottom panel). Shown are the means of three independent exper- iments done in duplicate for each construct. Bars indicate means+/−SD. *t-test, pb0.05. (B) Effect of TRAF2 knock-out on RIP1 levels. The endogenous protein levels of RIP1 was assessed by immunoblotting using a specific antibody in MEF WT and MEF TRAF2−/−.

Article Snippet: Mammalian expression vectors coding for human USP2a, human TRAF2 and human RIP1 were from Origene (USA).

Techniques: Transfection, Construct, Control, Staining, Knock-Out, Western Blot

Journal: Cells

Article Title: Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

doi: 10.3390/cells11030363

Figure Lengend Snippet: Figure 2. SOX9 silencing induces cancer cell death in vitro and in vivo. Knockdown of SOX9 in MDAH-2774 ovarian cancer cells increase anticancer drug responsiveness and inhibits tumorigenesis. (a) High expression of cytoplasmic SOX9 in MDAH-2774 was confirmed by immunocytochemistry. Alexa-488 (green) was used as a secondary antibody to visualize SOX9 protein expression. DAPI was used to stain the nucleus. (b and c) Stable knockdown of SOX9 mRNA and protein expression by lentivirus containing vector alone and SOX9-specific shRNAs (sh1 and sh2) was confirmed by qRT- PCR (b) and immunoblot analysis (c). (d) SOX9 silencing markedly altered the cell cycle, resulting in increased S phase and decreased G1 phase. (e) SOX9 silencing markedly induced cancer cell death in CDDP-resistant 2774 cells. (f) Inhibition of ovarian cancer cell proliferation by SOX9 silencing. Ovarian 2774 cell proliferation was analyzed by EZ-Cytox assay after a 72 h CDDP treatment. A significant reduction in cell viability by CDDP treatment in SOX9 knockdown cells. (g) Complete suppression of tumor formation by SOX9 knockdown. Stable 2774 cells harboring vector and SOX9-specific shRNA were injected into Balb/c mice, and tumor growth was analyzed for 35 days. Knockdown of SOX9 failed to form a detectable tumor mass. All data are presented as the mean ± SD of three independent experiments. ** p < 0.01, and *** p < 0.001 indicate a significant difference.

Article Snippet: RIPK1 small interfering RNAs (RIPK1 siRNA) (SR305755) were purchased from ORIGENE (Rockville, MD).

Techniques: In Vitro, In Vivo, Knockdown, Expressing, Immunocytochemistry, Staining, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Inhibition, shRNA, Injection

Journal: Cells

Article Title: Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

doi: 10.3390/cells11030363

Figure Lengend Snippet: Figure 3. Silencing of SOX9 in human ovarian 2774 cancer cells with high SOX9 expression resulted in loss of cancer stem-like properties. (a) SOX9 silencing strongly suppressed ALDH1A1 mRNA expression measured by qRT-PCR analysis. Control 2774 cells and 2774-Sox9sh cells harboring vector alone and SOX9- specific shRNA(sh1) were harvested to extract the total RNA for qRT-PCR assay to measure ALDH1A1 mRNA expression. (b) Reduction in ALDH activity by SOX9 knockdown. ALDH1 activity was analyzed by Aldefluor assay. (c) Spheroid formation was blocked by SOX9 knockdown in 2774 cells. Using 3D cell culture conditions, 2774-control cells formed discrete and compact spheres, whereas 2774-Sox9shRNA cells failed to form spheres. (d) Suppression of cancer cell invasion by SOX9 knockdown. Control vector 2774 and 2774-shSOX9 cells were seeded onto Transwells on Matrigel-coated Transwells. The number of invaded cells was counted under a light microscope, and mean values were plotted. Data present the mean ± SD of three independent experiments. *** p < 0.001 indicate significant difference.

Article Snippet: RIPK1 small interfering RNAs (RIPK1 siRNA) (SR305755) were purchased from ORIGENE (Rockville, MD).

Techniques: Expressing, Quantitative RT-PCR, Control, Plasmid Preparation, shRNA, Activity Assay, Knockdown, Cell Culture, Light Microscopy

Journal: Cells

Article Title: Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

doi: 10.3390/cells11030363

Figure Lengend Snippet: Figure 4. Stem cell factor SOX9 interacts with a cell death regulator RIPK1. (a) The amino acid sequence of human RIPK1 is indicated by single-letter abbreviations. The underlined amino acid sequence is the translated hRIPK1 protein identified from the yeast two-hybrid assay. (b) Identification

Article Snippet: RIPK1 small interfering RNAs (RIPK1 siRNA) (SR305755) were purchased from ORIGENE (Rockville, MD).

Techniques: Sequencing, Y2H Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB.

doi: 10.4161/cc.7.14.6310

Figure Lengend Snippet: Figure 3. The C-terminal domain of RIP1 activates NFκB. (A) Target sites of vector-based (sh24-26) and oligo-based (si7-10) RNAi are shown in aligned with the long and the short ripk1 transcripts. (B) Suppression of NFκB activity in 3B4.1 cells by anti-ripk1 shRNAs. The individual shRNAs were compared in an NFκB reporter assay by adding the respective expression vectors to the transfection mixture. Luciferase activity was normalized to that of the constitutive beta-galactosidase reporter. An shRNA against p53 was used as an off-target control and the corresponding luciferase activity was arbitrarily set at 100. (C) Suppression of NFκB activity in 3B4.1 cells, using transfec- tion of RNA oligos. After transfection with the indicated oligos, the next day the cells were transfected with the NFκB luciferase and beta-galactosidase reporter mixture. Reporter assay was done as in (B). Scrambled oligo was used as a control, and the respective luciferase activity was arbitrarily set as 100. (D) Activation of NFκB by expression of short RIP1 in naïve C6TA4 cells. Expression constructs for the putative short RIP1 and the cor- responding anti-sense RNA, or an empty vector control, were co-transfected with a constitutive beta-galactosidase vector and NFκB luciferase reporter. Luciferase activity, measured as in B, is displayed as fold over that observed in untreated vector-transfected cells.

Article Snippet: For vector-based RNAi, the plasmids that express shRNAs against RIPK1 (Origene) were added to the transfection mixture used for NFκB reporter assay.15 A vector encoding shRNA against p53 was obtained from Dr. Peter Chumakov (Cleveland Clinic).

Techniques: Plasmid Preparation, Activity Assay, Reporter Assay, Expressing, Transfection, Luciferase, shRNA, Control, Activation Assay, Construct

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB.

doi: 10.4161/cc.7.14.6310

Figure Lengend Snippet: Figure 7. (A) Northern analysis of RIP1 transcripts. RNA from C6TA4 cells and its mutant derivatives (3B4.1 and C4.1), with and without doxycycline treatment, as well as from the indicated tumor cell lines, was probed with a fragment corresponding to exons 7–9 of full-length ripk1. (B) Transcriptional activity of the putative internal promoter of the ripk1 gene in transient transfec- tion assays. For each of the four tested cell lines, luciferase activity was nor- malized to that of co-transfected constitutive beta-galactosidase reporter and expressed in relationship to the value for the promoter-less pGL3Basic vector. pRIPpromS and pRIPpromAS are derivatives of pGL3Basic with the putative promoter fragment in sense and anti-sense orientations, respectively.

Article Snippet: For vector-based RNAi, the plasmids that express shRNAs against RIPK1 (Origene) were added to the transfection mixture used for NFκB reporter assay.15 A vector encoding shRNA against p53 was obtained from Dr. Peter Chumakov (Cleveland Clinic).

Techniques: Northern Blot, Mutagenesis, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 1. RIPK1 is upregulated in human melanoma cells under ER stress induced by TM or TG. (A) Whole cell lysates from Mel-RM and MM200 cells with or without treatment with tunicamycin (TM) (3 mM) (left panel) or thapsigargin (TG) (1 mM) (right panel) for the indicated periods were subjected to western blot analysis of RIPK1, HSPA5, and GAPDH (as a loading control). The numbers represent fold changes of HSPA5. The data shown are representa- tive of 3 individual experiments. (B) Total RNA from Mel-RM and MM200 cells with or without treatment with TM (3 mM) (left panel) or TG (1 mM) (right panel) for the indicated periods were subjected to qPCR analysis of RIPK1 mRNA expression. The relative abundance of the RIPK1 mRNA before treatment was arbitrarily designated as 1 (n D 3, mean §SEM). (C) Total RNA from Mel-RM and MM200 cells treated with TM (3 mM) or TG (1 mM) for 16 h followed by treatment with actinomycin D (100 ng/ml) for the indicated period was subjected to qPCR analysis for the expression of RIPK1 mRNA. The relative abundance of the RIPK1 mRNA without actinomycin D treatment was arbitrarily designated as 1 (n D 3, mean §SEM, *P < 0.05, Student t test). (D) Whole cell lysates from ME4405, Sk-Mel-28, Mel-CV, IgR3 and Mel-RMu melanoma cells and HEMn-MP melanocytes treated with TM (3 mM) or TG (1 mM) for 16 h were subjected to western blot analysis of RIPK1, HSPA5, and GAPDH (as a loading control). The numbers represent fold changes of HSPA5. The data shown are representative of 3 individual experiments.

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Western Blot, Control, Expressing

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 2. RIPK1 protects melanoma cells from killing by TM or TG. (A) Whole cell lysates from Mel-RM and MM200 cells transduced with the control or RIPK1 shRNA treated with tunicamycin (TM) (3 mM) or thapsigargin (TG) (1 mM) for 16 h were subjected to western blot analysis of RIPK1, HSPA5, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. The numbers represent fold changes of HSPA5. (B) Mel-RM and MM200 cells transduced with the control or RIPK1 shRNA were treated with TM (3 mM) or TG (1 mM) for 48 h. Cell viability was measured by Cell- Titer-Glo assays (n D 3, mean §SEM, *P < 0.05, Student t test). (C) Mel-RM and MM200 cells transduced with the control or RIPK1 shRNA were seeded onto 6-well plates at 2000 cells per well in medium containing TM (1 mM) or TG (0.3 mM). Twelve d later, cells were fixed and stained with crystal violet. The data shown are representative of 3 individual experiments. (D) Mel-RM and MM200 cells stably transduced with the control or RIPK1 shRNA were transiently transfected with a shRNA-resistant mutant form of RIPK1 (pCMV-MYC-mut-RIPK1). Twenty-four h later, whole cell lysates were subjected to western blot analysis of RIPK1 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (E) Mel-RM and MM200 cells stably transduced with the control or RIPK1 shRNA were transiently transfected with a shRNA-resistant mutant form of RIPK1 (pCMV-MYC-mut- RIPK1). Twenty-four h later, cells were treated with TM (3 mM) or TG (1 mM) for another 48 h. Cell viability was measured by CellTiter-Glo assays (n D 3, mean §SEM, *P < 0.05; Student t test). (F) Whole cell lysates from HEMn-MP melanocytes transfected with the pCMV-MYC or pCMV-MYC-RIPK1 were sub- jected to western blot analysis of RIPK1 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (G) HEMn-MP melanocytes transfected with pCMV-MYC or pCMV-MYC-RIPK1 were treated with TM (3 mM) or TG (1 mM) for 48 h. Cell viability was measured by Cell- Titer-Glo assays (n D 3, mean §SEM, *P < 0.05, Student t test).

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Transduction, Control, shRNA, Western Blot, Staining, Stable Transfection, Transfection, Mutagenesis

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 5. RIPK1-mediated activation of MAPK8/9 contributes to induction of autophagy by ER stress in melanoma cells. (A) Mel-RM and MM200 cells transduced with the control or RIPK1 shRNA were treated with tunicamycin (TM) (3 mM) or thapsigargin (TG) (1 mM) for 16 h. Whole cell lysates were sub- jected to western blot analysis of RIPK1, pMAPK8/9, MAPK8/9, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (B) Whole cell lysates from Mel-RM and MM200 cells transfected with the control or MAPK8/9 siRNA were subjected to western blot analysis of MAPK8, MAPK9 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (C) Mel-RM and MM200 cells trans- fected with the control or MAPK8/9 siRNA were treated with TM (3 mM) or TG (1 mM) for 16 h. Whole cell lysates were subjected to western blot analysis of RIPK1, SQSTM1, MAP1LC3A, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (D) Mel-RM and MM200 cells were treated with TM (3 mM) or TG (1 mM) for 48 h with or without pretreatment with the MAPK8/9 inhibitor SP600125 (10 mM) for 1 h. Cell viability was measured by CellTiter-Glo assays (n D 3, mean §SEM, *P < 0.05, Student t test). (E) Mel-RM and MM200 cells transfected with the con- trol or MAPK8/9 siRNA 1 were treated with TM (3 mM) or TG (1 mM) for 48 h. Cell viability was measured by CellTiter-Glo assays (n D 3, mean §SEM, *P < 0.05, Student t test). (F) Mel-RMu cells stably transfected with pCMV-MYC or pCMV-MYC-RIPK1 were treated with TM (3 mM) or TG (1 mM) for 16 h with or without pretreatement with SP600125 (10 mM) for 1 h. Whole cell lysates were subjected to western blot analysis of pMAPK8/9, MAPK8/9, SQSTM1, MAP1LC3A, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (G) Whole cell lysates from Mel- RM cells treated with TM (3 mM) or TG (1 mM) for 16 h were immunoprecipitated by RIPK1 antibody. The resulting precipitates were subjected to western blot analysis of RIPK1 and MAPK8/9. The data shown are representative of 3 individual experiments.

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Activation Assay, Transduction, Control, shRNA, Western Blot, Transfection, Stable Transfection, Immunoprecipitation

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 7. XBP1 plays an important role in RIPK1 upregulation in melanoma cells upon ER stress. (A) Whole cell lysates from Mel-RM and MM200 cells transduced with the control, ERN1, ATF6, or EIF2AK3 shRNA were subjected to western blot analysis of ERN1 (left panel), ATF6 (middle panel) or EIF2AK3 (right panel) and GAPDH (as a loading control). The numbers represent knockdown efficiencies. The data shown are representative of 3 individual west- ern blot analyses. (B) Mel-RM and MM200 cells transduced with the control, ERN1, ATF6, or EIF2AK3 shRNA were treated with tunicamcyin (TM) (3 mM) or thapsigargin (TG) (1 mM) for 16 h. Whole cell lysates were subjected to western blot analysis of HSF1, RIPK1, SQSTM1, MAP1LC3A, and GAPDH (as a load- ing control). The data shown are representative of 3 individual experiments. (C) Mel-RM and MM200 cells transfected with the control or XBP1 siRNA were subjected to RT-PCR for the analysis of XBP1 mRNA. GAPDH was used as a loading control. The data shown are representative of 3 individual qPCR analyses. (D) Mel-RM and MM200 cells transfected with the control or XBP1 siRNA were treated with TM (3 mM) or TG (1 mM) for 16 h. Whole cell lysates were subjected to western blot analysis of HSF1, RIPK1, SQSTM1, MAP1LC3A, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments.

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Transduction, Control, shRNA, Western Blot, Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 8. Heat shock factor protein 1 (HSF1) is responsible for transcriptional upregulation of RIPK1 in melanoma cells upon ER stress. (A) Mel-RM (left panel) and MM200 (right panel) cells transfected with the indicated pGL3-basic based reporter constructs were treated with tunicamycin (TM) (3 mM) for 16 h followed by measurement of the luciferase activity (n D 3, mean §SEM, *P < 0.05, Student t test). (B) Formaldehyde-cross-linked chromatin of Mel- RM and MM200 with or without treatment with TM (3 mM) for 16 h was subjected to immunoprecipitation with antibody against HSF1 (top panel) or XBP1 (bottom panel). The precipitates were subjected to PCR amplification using primers for the ¡168 to C25 region of the RIPK1 promoter. The data shown are representative of 3 individual experiments. (C) Formaldehyde-cross-linked chromatin of Mel-RM and MM200 with or without treatment with TM (3 mM) for 16 h was subjected to immunoprecipitation with antibody against HSF1. The precipitates were subjected to PCR amplification using pri- mers for the HSP70-1 promoter. The data shown are representative of 3 individual experiments. (D) Mel-RM and MM200 cells transfected with indicated pGL3-basic based reporter constructs were treated with TM (3 mM) for 16 h followed by measurement of the luciferase activity (n D 3, mean §SEM, *P < 0.05, Student t test). (E) Whole cell lysates of Mel-RM and MM200 cells transfected with the control or HSF1 siRNA were subjected to western blot analysis of HSF1 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (F) Mel-RM and MM200 cells trans- fected with the control or HSF1 siRNA were treated with TM (3 mM) or thapsigargin (TG) (1 mM) for 16 h. Whole cell lysates were subjected to western blot analysis of RIPK1, SQSTM1, MAP1LC3A, pMAPK8/9, MAPK8/9, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (G) Mel-RM and MM200 cells transfected with the control or HSF1 siRNA were treated with TM (3 mM) or TG (1 mM) for 48 h. Cell viability was measured by CellTiter-Glo (n D 3, mean §SEM, *P < 0.05, Student t test).

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Transfection, Construct, Luciferase, Activity Assay, Immunoprecipitation, Control, Western Blot

Journal: Autophagy

Article Title: RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

doi: 10.1080/15548627.2015.1049800

Figure Lengend Snippet: Figure 9. Induction of autophagy by RIPK1 in melanoma cells upon pharmacological ER stress was due to activation of BECN1 as a result of its disassociation from BCL2L11. A schematic illustration of the proposed pharmacological ER stress-XBP1-HSF1-RIPK1-MAPK8/9-autophagy pathway.

Article Snippet: Transfection of siRNA was carried out as described previously.83 Short hairpin RNA (shRNA) knockdown Sigma-Aldrich MISSION Lentiviral Transduction Particles for shRNA-mediated knockdown of ERN1 (SHCLNVNM_001433), ATF6 (SHCLNV-NM_007348) and EIF2AK3 (SHCLNV-NM_032025) were purchased from Sigma-Aldrich and used as described previously.14 The human RIPK1 shRNA (TG320591), BCL2L11 shRNA (TG306422), BECN1 shRNA (TG314484) and scrambled shRNA (TG300130), were purchased from Origene, and were used to infect cells according to the manufacturer’s protocol.

Techniques: Activation Assay