Journal: The Journal of Biological Chemistry

Article Title: The long non-coding RNA HOTAIR enhances pancreatic cancer resistance to TNF-related apoptosis-inducing ligand

doi: 10.1074/jbc.M117.786830

Figure Lengend Snippet: Overexpression of HOTAIR attenuates TRA-8-induced apoptosis in sensitive pancreatic cancer cells. A and B, BxPC3 (A) and MiaPaCa-2 (B) cells were infected with lentiviruses carrying control vector or HOTAIR cDNA (HOTAIR), and stable clones were selected by puromycin. Panels Aa and Ba, HOTAIR expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, ***, p < 0.001). Panels Ab and Bb, TRA-8-induced apoptosis. BxPC3 and MiaPaCa-2 cells with HOTAIR overexpression and their control vector cells were seeded into 6-well plates at 2 × 105 per well. After culturing for 24 h, cells were exposed to TRA-8 (1 μg/ml) for 24 h, and apoptosis was determined by flow cytometry using Annexin PE and 7 AAD staining kit. TRA-8-induced apoptosis is shown in the hatched bars (n = 3, ***, p < 0.001). Panels Ac and Bc, Western blot analysis of the expression of caspase-8 (Casp8). The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown.

Article Snippet: Antibodies were purchased as follows: rabbit anti-DR5 antibody (ProSci, no. 2019, lot number 5355–1502), mouse anti-caspase 8 (BIOSOURCE no. AHZ0502, lot number 22363–01S), mouse anti-β-actin (Sigma, no. A5541–2MI, lot number 014M4759), rabbit anti-EZH2 (Cell Signaling Technology, no. D2C9, lot number 7), and mouse anti-trimethyl-histone H3 (lysine 27) (Active Motif, no. 61017, lot number 23115012).

Techniques: Over Expression, Infection, Plasmid Preparation, Clone Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Western Blot

Journal: Nature Communications

Article Title: The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

doi: 10.1038/ncomms10972

Figure Lengend Snippet: ( a ) MYC-MYO7A was co-expressed with V5-CASP8, V5-CASP9 or V5-CASP3 in 293T cells. α-MYC-immunoprecipitation was performed and MYO7A interaction was assessed via immunoblotting. ( b ) The indicated proteins were co-expressed in 293T cells and the interaction between MYO7A and full-length or deletion mutants of CASP8 was assessed by immunoprecipitation followed by immunoblotting. ( c ) Confocal microscopy images of SKOV3 cells showing co-localization of MYO7A (green) and CASP8 (red) using the indicated antibodies. Nuclei stained with DAPI (4,6-diamidino-2-phenylindole; blue). ( d ) Representative confocal microscopy images of proximity ligation assay (PLA) using α-MYO7A and either no α-CASP8 antibody (i) or rabbit α-CASP8 antibody (i–vii) in SKOV3 cells. PLA signals are in green and nuclei are stained with DAPI (blue). PLA between MYO7A and CASP8 on siRNA knockdown of control (non-targeting oligos) (iii) or RIPK1 (iv). Immunoblot analysis of RIPK1 and ACTIN following siRNA knockdown of control or Ripk1 . PLA between MYO7A and CASP8 in cells stably expressing CrmA (v), on treatment with 100 nM SM for 5 h (vi), or in response to SM/CrmA treatment (vii).

Article Snippet: The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Proximity Ligation Assay, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

doi: 10.1038/ncomms10972

Figure Lengend Snippet: ( a ) Proximity ligation assay (PLA) between RIPK1 and CASP8 in SKOV3 cells on control treatment (i), 100 nM SM for 5 h or in cells stably expressing CrmA (iii). ( b ) PLA between RIPK1 and CASP8 in HT1080 cells on treatment with zVAD (i) or TNF/SM/zVAD (ii). ( c ) PLA between RIPK1 and CASP8 in SWISS-3T3 cells on the indicated treatments. Nuclei are stained with DAPI (4,6-diamidino-2-phenylindole; blue), PLA signals are in green and Phalloidin staining is in red.

Article Snippet: The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000).

Techniques: Proximity Ligation Assay, Stable Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

doi: 10.1038/ncomms10972

Figure Lengend Snippet: ( a ) WB analysis of activated CASP8 (p43 and p18 cleavage products), MYO7A, RIPK1 or ACTIN following the indicated treatments in NIH-3T3 cells. Depicted are representative westernblot images of three biological repeats. ( b ) DEVDase assays from lysates of NIH-3T3 cells subjected to siRNA targeting Myo7A or Casp8 , and exposed to the indicated treatments. ( c ) FACS analysis of PI-positive NIH-3T3 or SWISS-3T3 cells subject to siRNA knockdown of the indicated genes followed by the indicated treatments. Error bars represent s.d. P -values are indicated. Experiments were conducted in triplicates.

Article Snippet: The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000).

Techniques:

Journal: Nature Communications

Article Title: The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

doi: 10.1038/ncomms10972

Figure Lengend Snippet: In Drosophila , binding of CK to DRONC favours DRONC-mediated cleavage and maturation of SGG46 into its active form SGG10. In mammals, MYO7A might regulate CASP8-mediated cleavage of RIPK1, which results in disassembly of the ripoptosome complex. In the absence of MYO7A, less RIPK1 would be cleaved, which results in a build-up of complex-II/ripoptosome formation, which in turn would lead to enhanced caspase activation.

Article Snippet: The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000).

Techniques: Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Cut-like homeobox 1 ( CUX1 ) tumor suppressor gene haploinsufficiency induces apoptosis evasion to sustain myeloid leukemia

doi: 10.1038/s41467-021-22750-8

Figure Lengend Snippet: a Cflar transcript expression by qRT-PCR in Cux1 +/− ; Flt3 ITD and Flt3 ITD Mac1 + cells. Expression was normalized to Gapdh . Biological and technical triplicates were analyzed. b Immunoblot showing increased CFLAR expression in whole bone marrow (top) and Mac1 + myeloid cells (bottom) from Flt3 ITD and Cux1 +/− ; Flt3 ITD mice. Vinculin was used as a loading control. The dashed line indicates splicing of non-adjacent lanes from the same blot. The experiment was performed twice. c Growth assays using 2 × 10 4 c-Kit + cells from two mice of the indicated genotypes plated in triplicate. Doxycycline (0.1 µg/ml, Dox) and/or Nec-1 (20 µM) was added and the number of live cells per well was determined by Annexin-V/DAPI staining 48 h later. d Annexin-V/DAPI staining to determine numbers of early- and late-apoptotic cells in doxycycline-treated cells from c . e Quantification of colonies derived from plating 500 c-Kit + cells transduced with doxycycline-inducible Cflar -targeting shRNA lentiviruses from indicated mice. Cells from two mice were plated in duplicate on cytokine-supplemented methylcellulose in the presence or absence of doxycycline. Colonies containing more than 50 cells were scored seven days after plating. f Immunoblot of lysates from Cux1 +/− ; Flt3 ITD c-Kit + cells transduced with doxycycline-inducible Cflar -targeting shRNA lentiviruses with and without 48 h of doxycycline treatment showing differences in caspase-8 and -3 activation. The immunoblot was performed once. g Immunoblot of lysates from Flt3 ITD c-Kit + cells showing effect of stable CFLAR expression on caspase-8 and -3 activation. The immunoblot was performed once. h Experimental outline of bone marrow transplant assay to assess selective impact of Cflar depletion on Cux1 +/− ; Flt3 ITD cells compared with control (Con), Cux1 +/− and Flt3 ITD cells. Hematopoietic progenitor cKit + cells from CD45.2 + mice of each genotype were infected with doxycycline-inducible Cflar -targeting shRNA lentiviruses and 10 5 puromycin-selected cells were transplanted with 10 5 wild-type CD45.1 + CD45.2 + bone marrow support cells into lethally irradiated CD45.1 + CD45.2 + recipient mice. After four weeks, mice were fed a doxycycline-containing diet to induce Cflar depletion or maintained on a normal diet. Donor-derived CD45.2 + bone marrow cells were quantified two weeks later by flow cytometry. i Quantification of donor-derived bone marrow CD45.2 + cells in recipient mice ( n = 6, per group) transplanted with control (Con), Cux1 +/− , Flt3 ITD or Cux1 +/− ; Flt3 ITD c-Kit + cells with and without doxycycline-induced Cflar depletion. j Spleen weights normalized to body weight of mice at the termination of the transplant. All plots show mean + /± s.e.m.; ns, not significant; two-way ( c ) or one-way ( d , j ) ANOVA with Tukey’s test for multiple comparisons, Two-tailed, unpaired t -test ( a , e , i ).

Article Snippet: The following primary antibodies used at 1:1,000 dilution were purchased from Cell Signaling Technology: anti-HDAC1 (5356); anti-CFLAR (56343); anti-β-Actin (3700); anti-CASP8 (9746); mouse-specific anti-CASP8 (4927); anti-cleaved CASP8 (9496); mouse-specific anti-cleaved CASP8 (8592); anti-PARP (9542); anti-CASP3 (9662); anti-cleaved CASP3 (9661); anti-cleaved PARP (5625); anti-MLKL (37705); anti-p-MLKL S345 (37333); anti-XIAP (2042); anti-NFκB p65 (8242); anti- IκBα (4814); anti-NFκB p100/p52 (4882); and anti-vinculin (13901).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining, Derivative Assay, Transduction, shRNA, Activation Assay, Infection, Irradiation, Flow Cytometry, Two Tailed Test