Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Representative flow cytometry plots of NK92 cells transduced with AAV-SB-HER2.CAR virus (MOI = 1 × 104 and 1 × 105), transduced with HER2.CAR lentivirus (MOI = 1 and 2.5), or electroporated with plasmid DNA (2 μg = 1 μg transposon plasmid + 1 μg transposase plasmid) at three different time points. b, Quantification of a. c, Flow-cytometry measurement of HER2.CAR expression in NK92 cells on day 33 after mRNA electroporation and AAV-SB-HER2.CAR viral transduction. d, Quantification of c. e, Cytolysis analysis of MCF7-PL (MCF7 with puromycin resistance and luciferase expression) cancer cells that were co-cultured with NK92-AAV-SBHER2.CAR cells. CAR-NKs were seeded at two E:T ratios, and a luciferase assay was performed at two time points (24 h and 48 h). Error bars, mean ± s.e.m. of three biological replicates.

Article Snippet: HEK293T, NALM6, MM.1R, MCF7, NK-92, THP-1, human CD14 + monocytes, human peripheral blood mononuclear cells (PBMCs) and human iPSCs were purchased from commercial sources (ThermoFisher, American Type Culture Collection and StemCell).

Techniques: Flow Cytometry, Transduction, Virus, Plasmid Preparation, Expressing, Electroporation, Luciferase, Cell Culture

Journal: Nature communications

Article Title: Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

doi: 10.1038/ncomms4361

Figure Lengend Snippet: ( a ) MCF-10A cells were infected by the indicated lentiviral particles targeting the indicated genes. Control cells and knockdown cells were subjected to microarray analysis. The presence of the HRD gene signature was analyzed by supervised clustering analysis. ( b ) Modified HR repair assay was performed in MCF-10A cells by transfecting cells with DRGFP DSB substrate plasmid and I-SceI plasmid through electroporation, followed by analysis of GFP-positive cells by flow cytometry 48 to 72 hours later. Student’s t -test was performed from results of three independent experiments as mean + SD. ( c ) The rate of cell survival in response to olaparib was determined by colony formation assay. Each value was relative to control cells without treatment and represents the mean ± SD from three independent experiments. Student’s t -test showed that treatment response differed between BRCA1-PTEN double knockdown cells and single knockdown cells (P<0.001). ( d ) Heat map of the HRD gene signature with the 26 genes most significantly changed in BRCA1-PTEN double knockdown cells compared to single-knockdown-cells. ( e ) Microarray data from 295 breast cancers were clustered into basal-like, Her2-positive (Her2), luminal A, luminal B, and normal breast-like. Gene expression levels of TTK among the different breast cancer subtypes are indicated. ( f ) Quantitative analysis of HR repair assay in cells transfected with TTK plasmids. Results are shown as mean + SD from three independent experiments. Student’s t -test showed that over-expression of TTK significantly increased HR repair efficiency (P<0.05). BRCA1 SMARTpool siRNA was used as a positive control. Western blots demonstrating effective knockdown are shown to the bottom.

Article Snippet: BRCA1 (D-9) monoclonal and TTK polyclonal antibodies were purchased from Santa Cruz (SC-6954, 1:1000) and Cell Signaling (#3255, 1:1000), respectively.

Techniques: Infection, Control, Knockdown, Microarray, Modification, Plasmid Preparation, Electroporation, Flow Cytometry, Colony Assay, Gene Expression, Transfection, Over Expression, Positive Control, Western Blot

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Competition-based proliferation assays in 23 human cancer cell lines infected with the indicated sgRNAs. PDAC, pancreatic ductal adenocarcinoma; RMS, rhabdomyosarcoma. n = 3. (B) Western blot of whole-cell lysates from MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. (C) Competition-based proliferation assay in MOLM-13 cells infected with the indicated sgRNAs. n = 3. (D) Western blot of whole-cell lysates from K562 cells on day 5 post-infection with the indicated sgRNAs. (E) Competition-based proliferation assay in K562 cells infected with the indicated sgRNAs. n = 3. (F) Quantification of the different cell-cycle stages in MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. n = 3. (G) Bioluminescence imaging of NSG mice transplanted with luciferase + /Cas9 + MOLM-13 cells infected with either sgROSA or sgSCP4. (H) Quantification of bioluminescence intensity. n = 3. p value was calculated by unpaired Student’s t-test. *p < 0.05. (I) Western blot of whole-cell lysates from CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. (J) qRT-PCR analysis of indels presence in CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs over the course of culturing in myeloid conditions. The effects of individual sgRNAs for SCP4 were averaged. n = 4. (K) Quantification of the flow cytometry analysis of myeloid differentiation of CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. The effects of individual negative controls and sgRNAs for SCP4 were averaged. n = 4. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines. “e” refers to the exon number targeted by each sgRNA. The indicated sgRNAs were linked to GFP. ROSA, Mock, and NT, negative controls; PCNA and MYC, positive controls. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Infection, Western Blot, Proliferation Assay, Imaging, Luciferase, Electroporation, Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Relative evolutionary conservation score for each residue from 0 — least conserved to 1 — most conserved. Based on data from . (B) Protein disorder prediction score for each residue from 0 — least disordered to 1 — most disordered. Based on data from . (C) Running average of log 2 fold changes of the CRISPR scan of SCP4 with all the possible sgRNAs. SCP4 protein amino acid numbers are indicated along the x axis. (D) Domain architectures of human SCP4 and the truncated version of SCP4 used in this study. (E) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 . (F) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 infected with the indicated sgRNAs. n = 3. (G) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector (EV; underloaded) or CRISPR-resistant wild type (WT) and catalytic mutants of SCP4. Vertical black dashed lines indicate omitted lanes from the same gel and same exposure. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant WT and catalytic mutants of SCP4 infected with the indicated sgRNAs. n = 3. All bar graphs represent the mean ± SEM. ROSA, negative control; PCNA, positive control. See also and .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Residue, CRISPR, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Proliferation Assay, Infection, Negative Control, Positive Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Western blot of cytoplasm and nuclear fractions from MOLM-13 cells. (B) Representative FLAG-SCP4 affinity purification western blot analysis for the subsequent mass spectrometry (MS) analysis. Cytoplasm and nucleus fractions from MOLM-13 cells stably expressing empty vector, FLAG-SCP4 WT, or catalytic mutant (D293A). Nuclear fraction was mixed with anti-FLAG M2 agarose overnight. The flow-through was analyzed to ensure efficient binding of the FLAG-tagged constructs (loaded as “unbound”). Following extensive washing, the agarose amount equivalent to the cytoplasm and nucleus fractions loading was boiled in Laemmli buffer and loaded as “FLAG-IP.” The rest was sent for the MS analysis. (C) Venn diagram depicting the overlap between proteins detected by MS and absent in empty vector controls in the 2 independent biological replicates. (D) Total unique peptide counts for SCP4, PDIK1L, and STK35 detected by MS in the 2 independent biological replicates. (E) Domain architectures and homology heat-map of human STK35 and PDIK1L. ATP-BS, ATP-binding site. (F–H) Immunoprecipitation followed by western blotting performed with the indicated antibodies. The whole-cell lysate was prepared from HEK293T 24 h post-transfection with the indicated constructs (F). The nuclear lysates were prepared from the human MOLM-13 cells stably expressing the indicated constructs (G and H). –, empty vector; WT, wild-type FLAG-SCP4; 236–466, FLAG-SCP4 236–466 ; D293A, catalytic mutant FLAG-SCP4 D293A ; IP, immunoprecipitation. Note: degradation bands appear in the WT and D293A input at ~50 kDa and in D293A at ~40 kDa. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Immunoprecipitation, Transfection

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A and B) Western blot of cytoplasm (Cyto) and nucleus (Nucl) fractions of MOLM-13 cells stably expressing HA-PDIK1L (A) or HA-STK35 (B) on day 5 post-infection with the indicated sgRNAs. Shown is a representative experiment of 3 independent biological replicates. (C) Schematics of phosphorylation sites reported in the publicly available phospho-proteomics datasets on STK35 and PDK1L relative to their domain architectures ( ; ). aa #, amino acid number; P, phosphorylation; ATP-BS, ATP-binding site. (D) Phosphorylated peptides assayed for SCP4 phosphatase activity in vitro . (E) Recombinant SCP4 protein purity as assessed by SDS-PAGE and Coomassie blue staining. His-SUMO-(TEV)-SCP4 was expressed in BL21 (DE3) cells and purified by affinity, anion exchange, and gel filtration chromatography. Molecular weight markers are shown for reference. (F and G) Phosphatase activity of SCP4 against the indicated peptides plotted for kinetic fitting. Each measurement was conducted in triplicate with standard deviations shown as error bars. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing EV or the CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions infected with the indicated sgRNAs. n = 3. (I) Western blot of whole-cell lysates from MOLM-13 cells stably expressing EV or CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions. All bar graphs represent the mean ± SEM. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Stable Transfection, Expressing, Infection, Phospho-proteomics, Binding Assay, Activity Assay, In Vitro, Recombinant, SDS Page, Staining, Purification, Filtration, Chromatography, Molecular Weight, Proliferation Assay, Construct

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Venn diagram depicting the overlap between statistically significant downregulated genes in MOLM-13 cells upon SCP4 knockout and STK35/PDIK1L double knockout. DeSeq2 (n = 4). (B) Ontology analysis of overlapping statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout. (C) Selected statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout and the amino acids they are involved in biosynthesis or transport of. (D) The correlation between log2 fold changes in the levels of selected metabolites upon SCP4 knockout and STK35/PDIK1L double knockout relative to negative control as measured by the MS analysis. Every dot represents the mean ± SEM (n = 6). Amino acids are in red, and there are a few unchanged metabolites in black for reference. Shown is a representative experiment of 3 independent biological replicates. (E) Model. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Knock-Out, Double Knockout, Negative Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Virus, Recombinant, Lysis, Plasmid Preparation, Magnetic Beads, Cloning, Purification, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Gel Extraction, Ligation, Sample Prep, Mass Spectrometry, Software

Journal: Stem cell research

Article Title: Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9

doi: 10.1016/j.scr.2021.102554

Figure Lengend Snippet: Generation and characterization of patient and gene corrected NGLY1 (c.1201 T > A) lines (NCATS-CL6103, NCATS-CL6104, NCATS-CL6105). (A) Schematic of editing approach, the mutant residue is highlighted in red with the desired edit in green. The sgRNA recognizes the mutant target sequence inducing a cut before the C residue before the PAM. The donor template then guides the repair of the sequence with the desired edit (T > A) in addition to a silent mutation (A > G) highlighted in gray. (B) Sequencing of PCR amplicons confirming presence either the mutant allele or heterozygous and homozygous correction and a silent homozygous mutation. (C) PCR result showing the clearance of Sendai virus and exogenous reprogramming factors in 592D iPSCs. (D) Phase contrast and immunofluorescence images showing pluripotent phenotype and the expression of pluripotency markers (NANOG, SOX2, OCT4 and SSEA4). (E) Flow cytometry data showing the percentage of cells expressing pluripotency markers (Tra-1–60 and NANOG). (F) Teratoma formation demonstrating the normal ectodermal, mesodermal and endodermal differentiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Pluripotency Markers , Rabbit anti-NANOG , 1:400 , Cell signaling, Cat# 4903, RRID: AB_10559205.

Techniques: Mutagenesis, Residue, Sequencing, Virus, Immunofluorescence, Expressing, Flow Cytometry

Journal: Stem cell research

Article Title: Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9

doi: 10.1016/j.scr.2021.102554

Figure Lengend Snippet: Reagents details

Article Snippet: Pluripotency Markers , Rabbit anti-NANOG , 1:400 , Cell signaling, Cat# 4903, RRID: AB_10559205.

Techniques: Immunocytochemistry, Flow Cytometry, Expressing, CRISPR, Electroporation, Selection

Journal: Stem cell research

Article Title: Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9

doi: 10.1016/j.scr.2021.102554

Figure Lengend Snippet: Characterization and validation

Article Snippet: Pluripotency Markers , Rabbit anti-NANOG , 1:400 , Cell signaling, Cat# 4903, RRID: AB_10559205.

Techniques: Immunocytochemistry, Flow Cytometry, Plasmid Preparation, Modification, Mutagenesis, Sequencing

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A H&E staining and expression of Ki-67 and N-GSDMD in normal skin tissue samples and skin lesion samples from psoriasis patients were shown. Red arrow indicates N-GSDMD. B Cleaved caspase-1 (green) and PI staining (red) were visualized by immunofluorescence assay. Nucleus was stained by Hoechst ( n = 10). Red arrow indicates PI positive cells. C The cleavage levels of GSDMD and IL-1β were detected in epidermis lysate of normal skin tissue samples and skin lesion samples from psoriasis patients through western blotting assay. D WT mice (C57BL/6NGpt) were stimulated by imiquimod or vaseline, respectively. Skin appearances were presented. Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67. The severity of the lesions was evaluated by PASI scores. The epidermal thickness was measured by Image J software. n = 4. E The co-location of GSDMD and cellular membrane was presented by immunofluorescence assay. Nucleus was stained by Hoechst. Actin filaments was stained by phalloidin ( n = 4). F The cleavage levels of caspase-1, GSDMD and IL-1β were detected in epidermis lysate of mice through western blotting assay. G Flow cytometry was used to determine the level of keratinocyte death. Scale bar represents 100 μm in A . Scale bar represents 500 μm in B . Scale bar represents 50 μm in E . * p < 0.05, ** p < 0.01, *** p < 0.001, WT wild type, GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Expressing, Immunofluorescence, Western Blot, Immunohistochemistry, Software, Membrane, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A – F WT (C57BL/6NGpt) and Gsdmd −/− mice were stimulated by imiquimod or vaseline, respectively. A Skin appearances were presented. B The severity of the lesions was evaluated by PASI scores (for each group, n = 6). C The epidermal thickness was measured by Image J software (for each group, n = 6). D Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67 ( n = 6). E The levels of loricrin, K5 and MPO were detected by immunohistochemistry study ( n = 6). F Western blotting assay was used to detect the level of cleaved GSDMD and cleaved IL-1β in epidermis lysate of WT or Gsdmd −/− mice ( n = 3). G WT and Gsdmd −/− mice were intradermally injected with recombinant mouse IL-23 (1 μg) in the right ear. The left ear was intradermally injected with vehicle. Skin appearances after injection were presented. H The severity of the lesions was evaluated by PASI scores (for each group, n = 4). I The epidermal thickness was measured by Image J software (for each group, n = 4). J Histological features were analyzed by H&E staining. Scale bar represents 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Software, Staining, Immunohistochemistry, Western Blot, Injection, Recombinant

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: Control (Krt14 +/+ - Gsdmd flox/flox ) mice and Gsdmd cko (Krt14 Cre/+ - Gsdmd flox/flox ) mice were stimulated by imiquimod or vaseline ( A – H ). A Skin appearances were presented. B The severity of the lesions was evaluated by PASI scores (for each group, n = 5). C The epidermal thickness was measured by ImageJ software (for each group, n = 5). D Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67 ( n = 5). E The efficiency of keratinocyte-specific GSDMD knockout was confirmed by immunofluorescence assay. F Western blotting assay was used to detect the level of cleaved GSDMD and cleaved IL-1β in epidermis lysate of control or Gsdmd cKO mice. G The levels of IL-17 and TNF-α in peripheral blood of imiquimod-stimulated control mice or Gsdmd cKO mice were measured by ELISA ( n = 3). H Ki-67+ and FVS 780+ epidermal cells from imiquimod-stimulated control mice or Gsdmd cKO mice were measured by flowcytometry ( n = 3). Scale bar represents 100 μm in D . Scale bar represents 50 μm in E . * p < 0.05, ** p < 0.01, *** p < 0.001. GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Software, Staining, Immunohistochemistry, Knock-Out, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A GSDMD siRNA or nonsense siRNA was transfected by electroporation into primary human epidermal keratinocytes. The efficiency of knockdown was measured by western blotting assay. siRNA 342 was chosen in the experiments afterwards. B Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). C PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay ( n = 4). D – F Keratinocytes were treated by M5 for 24 h in presence or absence of DSF (40 μM). D Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). E PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay ( n = 9). F The secretion of IL-1β, CXCL-2, CCL-20, IL-8 and S100A8/A9 were determined by ELISA ( n = 3). G Keratinocytes were treated by M5 for 24 h in presence or absence of Cu(DTC) 2 . Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). H , I Imiquimod-induced psoriasis-like dermatitis mice were topically applicated by 1%, 2%, 5% DSF or vehicle. H Skin appearances were presented. Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67. n = 3. I The cleavages of caspase-1, GSDMD and IL-1β in epidermis lysate were detected by western blotting assay ( n = 3). NC: nonsense. Scale bar represents 100 μm. Scale bar represents 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, DSF disulfiram, GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD, siNC nonsense control siRNA, siGSDMD GSDMD siRNA.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Electroporation, Knockdown, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Control

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: ( A ) Ex vivo primary murine splenic B cells were treated with 100 μM CK-689 or CK-666 for 1 hr. SPT was then carried out as in , using Cy3-labeled Fab fragments of antibodies to CD19. Single-particle trajectories from a representative cell are plotted using a color-coded temporal scale (left panels). Scale bars: 5 µm. Diffusion coefficients (center panels) and the diameter of maximum displacement (confinement diameter, right panels) over the 10 s period of observation were calculated for each track and cumulative frequency curves are shown. The dots on the curves indicate the median values. ****p<0.0001; Kolmogorov-Smirnov test. ( B–E ) Primary murine B cells were pre-treated with 100 μM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Ig κ surrogate Ag. Cells were fixed at the indicated time points and stained with an antibody that recognizes the surrogate Ag and with an antibody that recognizes phosphorylated CD19. Representative cells are shown ( B ). Scale bars: 2 µm. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. Beeswarm plots in which each dot represents one cell are plotted with the median (red line) and interquartile ranges (red box) for >125 cells per time point from a representative experiment ( C ). For each cell in ( C ), the fraction of total pCD19 fluorescence that overlaps with BCR-Ag microclusters was quantified by calculating the Manders’ coefficient ( D ). For each cell in ( C ), the total fluorescence intensity of pCD19 that was within BCR-Ag microclusters in cells was quantified ( E ). ****p<0.0001; ***p<0.001; ns, not significant; Mann-Whitney U test.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Ex Vivo, Labeling, Single Particle, Diffusion-based Assay, Expressing, Staining, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: ( A ) Primary murine B cells were treated with CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the anti-Ig κ surrogate Ag. The cells were fixed at indicated times and stained for surrogate Ag (anti-Ig κ ) and pCD19. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. For each condition, the median pCD19 fluorescence intensity was determined for >15 cells per experiment. For each experiment, the median pCD19 fluorescence intensity for CK-666-treated cells was expressed as a percent of the median value for CK-689-treated cells (=100%). This ratio is plotted for four independent experiments. ( B ) Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then stimulated with 10 µg/ml soluble anti-Ig κ for the indicated times. pCD19 and total CD79a (loading control) immunoblots are shown (left panels) and the pCD19/total CD79a ratios are graphed (right panels). Dotted red line corresponds to the pCD19/total CD79a ratio value for unstimulated CK-689-treated B cells. Representative data from one of three experiments. See for full blots. ( C ) The co-localization of pSyk with BCR-Ag clusters is not dependent on Arp2/3 complex activity. The co-localization of pSyk and Ag in the cells that were analyzed in was quantified using Manders’ coefficient.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Expressing, Staining, Fluorescence, Control, Western Blot, Activity Assay

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: Images of blots that were cropped for presentation in , , and are shown. The portions of the blots that were shown in the indicated figures are outlined by a red dashed box. Molecular weight markers are shown in kDa. ( A ) Full blots for and . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with COS-7 APCs expressing anti-Ig κ (left) or with soluble anti-Ig κ (right) for 0, 3, 5, 15 or 30 min. The upper blots were probed with anti-pCD79 antibodies and the lower blots with anti-CD79a antibodies. ( B ) Full blots for , an additional independent experiment carried out as in ( A ). ( C ) Full blots for . Primary murine splenic B cells were treated with DMSO (lane 1), CK-689 (lane 2), CK-666 (lane 3) for 1 hr, or stimulated with anti-Ig κ antibodies for 5 min (lane 4). The blots were probed with anti-pAkt plus anti-pERK antibodies (upper blot) or with anti-ERK plus anti-Akt antibodies (lower blot). ( D ) Full blots for . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with soluble anti-IgΚ for 0, 3, 5, 15 or 30 min. The left blot was probed with anti-pCD19 antibodies and the right blot with anti-CD79a antibodies as a loading control.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Molecular Weight, Expressing, Control

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet:

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Sequencing, Immunofluorescence, Western Blot, Single-particle Tracking, Flow Cytometry, Labeling, Cell Isolation, Electroporation, Recombinant, Software

Journal: Nature biomedical engineering

Article Title: Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone.

doi: 10.1038/s41551-019-0502-4

Figure Lengend Snippet: Fig. 1 | sEVs contain few miRNAs and are enriched in miR-451 compared with cells. a, Western blot of equal amounts of proteins from cells and the sEVs they produce for markers of sEVs (Tsg101 and flotillin 2), proteins excluded from sEVs (mitochondrial Tomm20) and Ago2. b, Nanoparticle tracking analysis of sEVs. Representative plot of particle number (y axis) versus diameter in nm (x axis) of sEV preparations from NSC-34 cells. c, Left, RT–qPCR of SOD1 siRNA recovered by ultracentrifugation after electroporation with or without sEVs. Unpaired two-tailed t-test; n = 3 for all groups. Right, RT–qPCR of SOD1 siRNA recovered by ultracentrifugation and then treated with RNase A and RNase T1 after electroporation with or without sEVs (NSC-34). Repeated measurements one-way analysis of variance (ANOVA) with Holm–Sidak correction; n = 3. d, Absolute copy numbers of miRNAs in sEVs measured by standard curve RT–qPCR analysis in sEVs produced by three cell types (MDA-MB-231 (n = 3), MEF (n = 2) and NSC-34 (n = 3)). e, Enrichment of miRNAs in sEVs, expressed as the level of the respective miRNA in total sEV RNA relative to its level in total cell RNA as measured by RT–qPCR, normalized to miR-16. MEF (n = 2) and NSC-34 (n = 3)). f, Enrichment of miR-451 in sEVs versus its level in cells in the listed cell types. n = 6 (mesenchymal stem cell (MSC) and macrophage) and n = 3 (all others). g, RT–qPCR to measure relative quantity of miR-451 in sEVs (NSC-34) left untreated, treated with RNase or treated with detergent and RNase. n = 2. h, Sucrose density gradient fractionation of sEVs produced by NSC-34 cells. Top: densities of fractions measured by refractometry (black line) and quantity of miR-451 (red dots, RT–qPCR, 2 ct without normalization) in these fractions. Western blot for Ago2 was performed on a separate membrane with the same fractions as western blots for Alix and flotillin 2. i, RT–qPCR analysis of combined fractions 6 and 7 from h either left untreated, treated with RNase A and RNase T1, or RNase A/T1 and 0.5% Triton X-100. Full blot images from a and h are presented in Supplementary Information. Data are mean ± s.e.m. NS, not significant.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibody (clone) Company Catalog # Alix (3A9) Cell Signaling Technology 2171S Ago2 (C34C6) Cell Signaling Technology 2897S B-Actin (AC-15) Sigma-Aldrich A5441 Flotillin2 (C42A3) Cell Signaling Technology 3436S GFP (GF28R) eBiosciences 14-6674-82 Tomm20 (FL-145) Santa-Cruz Biotechnology sc-11415 TSG101 (4A10) GeneTex GTX70255 TTR (Polyclonal) ThermoFisher PA5-80197 Tubulin (DM1A) Sigma-Aldrich T6199 Anti-Mouse HRP Jackson ImmunoResearch 115-035-174 Ant-Rabbit HRP Jackson ImmunoResearch 111-035-144 Validation Antibodies were used with species recommended by the manufacturers.

Techniques: Western Blot, Quantitative RT-PCR, Electroporation, Two Tailed Test, Produced, Fractionation, Membrane

Journal: Nature biomedical engineering

Article Title: Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone.

doi: 10.1038/s41551-019-0502-4

Figure Lengend Snippet: Fig. 2 | Reprogramming the pre-miR-451 backbone with siRNAs causes their enrichment in sEVs. a, Schematic of the predicted pre-miR-451 secondary structure, detailing its cleavage by Drosha, Ago2 and subsequent trimming by exonucleases. b, Northern blot of GFP siRNA produced from the pre-miR-451 backbone in HeLa and NSC-34 cells. c,d, RT–qPCR measuring enrichment of TetR (c) or GFP (d) siRNA in sEVs versus their levels in cells when these were integrated in either the pre-miR-451 backbone or the pre-miR-16 backbone (control) and transiently expressed in MEF. Two-tailed unpaired t-test; n = 3. e, RT–qPCR measuring enrichment of SOD1 siRNA in sEVs versus the level in cells when SOD1 siRNA was integrated in either the pre-miR-451 backbone or the pre-miR-16 backbone (control) and transfected into the indicated cell types. One-way ANOVA with Holm–Sidak correction; n = 4, 3, 2 and 6 for BV2, C8D1A, C8S and NSC-34 pre-miR-16 control transfected, respectively; n = 4, 3, 3, and 5 for the same cell types, respectively, transfected with pre-miR-451. f, Schematic portraying the reprogramming of the pre-miR-451 hairpin structure with siRNA targeting SOD1 or GFP siRNA. g, Copy number of GFP or SOD1 siRNA in sEVs after stable expression from the pre-miR-451 backbone in the indicated cell types. n = 3. h, Northern blot of GFP siRNA or U6 (control) in equal amounts of RNA from cells or sEVs from MEFs. i, RT–qPCR analysis of GFP siRNA processed integrated in the pre-miR-451 backbone in fractions of density gradient in Fig. 1k using the 2 ct method without normalization. j, Western blot of Ago2 and tubulin (loading control) in WT, Ago2−/− or Ago2−/− cells stably rescued with Ago2. Right: northern blot of GFP siRNA programmed into the pre-miR-451 backbone or U6 (loading control) in cellular RNA of Ago2−/− cells or Ago2−/− cells stably rescued with Ago2. Intermed., intermediate. k,l, RT–qPCR of miR-16 and miR-451 (k) or SOD1 siRNA integrated in the pre-miR-451 hairpin structure (l) in sEVs versus cells, using Ago2−/− or Ago2−/− cells stably rescued with Ago2. Enrichment in sEVs versus cells normalized to U6 (k) or miR-16 (l) RNA. n = 3. m, RT–qPCR to measure fold change in enrichment in sEVs versus cells of miR-451 after transiently expressing GFP (control) or Ago2. Full blot images from b, g and k are presented in the Supplementary Information. One-way ANOVA with Holm–Sidak multiple comparison, unless otherwise mentioned. Data are mean ± s.e.m.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibody (clone) Company Catalog # Alix (3A9) Cell Signaling Technology 2171S Ago2 (C34C6) Cell Signaling Technology 2897S B-Actin (AC-15) Sigma-Aldrich A5441 Flotillin2 (C42A3) Cell Signaling Technology 3436S GFP (GF28R) eBiosciences 14-6674-82 Tomm20 (FL-145) Santa-Cruz Biotechnology sc-11415 TSG101 (4A10) GeneTex GTX70255 TTR (Polyclonal) ThermoFisher PA5-80197 Tubulin (DM1A) Sigma-Aldrich T6199 Anti-Mouse HRP Jackson ImmunoResearch 115-035-174 Ant-Rabbit HRP Jackson ImmunoResearch 111-035-144 Validation Antibodies were used with species recommended by the manufacturers.

Techniques: Northern Blot, Produced, Quantitative RT-PCR, Control, Two Tailed Test, Transfection, Expressing, Western Blot, Stable Transfection, Comparison

Journal: Oncotarget

Article Title: STAT3 is constitutively acetylated on lysine 685 residues in chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.26110

Figure Lengend Snippet: ( A ) Cell lysates of cells from 16 patients with CLL were analyzed by Western immunoblotting using acetyl-STAT3, serine pSTAT3 and total STAT3 antibodies. Set2 cells were used as positive controls and GAPDH as loading control. ( B ) Low density PB cells were double stained for CD19 and acetyl-STAT3 antibodies and their corresponding isotype antibodies. In CLL cells of this patient, 74.6% of the cells stained positively for both CD19 and acetyl-STAT3. A representative experiment from 5 experiments that were conducted using PB samples from 5 different patients is depicted. ( C ) Acetyl-STAT3 and serine pSTAT3 co-immunoprecipitated with STAT3 antibodies. CLL cell lysates from 8 patients were immunoprecipitated with anti-STAT3 antibodies. The immune complex was then separated using SDS-PAGE. In all patient samples, serine pSTAT3 and acetyl-STAT3 were readily detected. HEK 293 cells were used as a positive control. Total STAT3 antibodies were used as loading control. I.P., Immunoprecipitate; B, beads coated with STAT3 isotype antibodies.

Article Snippet: After blocking, the membrane was incubated with the following primary antibodies: monoclonal mouse anti-human STAT3 (BD Bioscience, Palo Alto, CA, USA), monoclonal mouse anti-human Ser 727 STAT3 (Cell Signaling, Danvers, MA, USA), Rabbit anti-human acetyl STAT3 (Lys 685) antibodies (Cell Signaling), mouse anti-human p300 antibodies (Pierce Biotechnology Inc./Thermo scientific, Rockville, IL), and mouse anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Control, Staining, Immunoprecipitation, SDS Page, Positive Control

Journal: Oncotarget

Article Title: STAT3 is constitutively acetylated on lysine 685 residues in chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.26110

Figure Lengend Snippet: ( A ) Low density PB cells from 4 patients were stained with CD19, serine pSTAT3 and acetyl-STAT3 antibodies and analyzed using flow cytometry. The rate of cells expressing CD19 was 87%, of serine pSTAT3 was 53%, of cells expressing acetyl-STAT3 was 55.9%, and of cells expressing CD19, serine pSTAT3 and acetyl-STAT3 was 26.9%. ( B ) CLL cell protein from PB samples of 7 patients was immunoprecipitated with anti-serine pSTAT3 antbodies. As shown, acetyl-STAT3 was detected in the serine pSTAT3 immunoprecipitate of all patients. Jurkat cells were used as a positive control. I.P., Immunoprecipitate; B, beads coated with serine pSTAT3 isotype antibodies.

Article Snippet: After blocking, the membrane was incubated with the following primary antibodies: monoclonal mouse anti-human STAT3 (BD Bioscience, Palo Alto, CA, USA), monoclonal mouse anti-human Ser 727 STAT3 (Cell Signaling, Danvers, MA, USA), Rabbit anti-human acetyl STAT3 (Lys 685) antibodies (Cell Signaling), mouse anti-human p300 antibodies (Pierce Biotechnology Inc./Thermo scientific, Rockville, IL), and mouse anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Staining, Flow Cytometry, Expressing, Immunoprecipitation, Positive Control

Journal: Oncotarget

Article Title: STAT3 is constitutively acetylated on lysine 685 residues in chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.26110

Figure Lengend Snippet: ( A ) Western immunoblotting showing increased levels of p300 in 4 patients and low levels in one patient with CLL but not in CD19+ B cells from 2 healthy volunteers. HELA and Jurkat cells were used as controls. ( B ) CLL cells were transfected with p300-siRNA or GAPDH using electroporation. As shown in the left panel, qRT-PCR analysis showed that transfection with p300-siRNA downregulated p300 transcript levels by approximately 7-fold. A Western blot analysis depicted in the right panel showed that transfection of CLL cells with p300-siRNA downregulated p300, acetyl-STAT3, serine pSTAT3, and STAT3 protein levels. A representative of 4 experiment using samples of 4 different patients yielded similar results. A representative experiment is depicted.

Article Snippet: After blocking, the membrane was incubated with the following primary antibodies: monoclonal mouse anti-human STAT3 (BD Bioscience, Palo Alto, CA, USA), monoclonal mouse anti-human Ser 727 STAT3 (Cell Signaling, Danvers, MA, USA), Rabbit anti-human acetyl STAT3 (Lys 685) antibodies (Cell Signaling), mouse anti-human p300 antibodies (Pierce Biotechnology Inc./Thermo scientific, Rockville, IL), and mouse anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Transfection, Electroporation, Quantitative RT-PCR

Journal: Oncotarget

Article Title: STAT3 is constitutively acetylated on lysine 685 residues in chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.26110

Figure Lengend Snippet: ( A ) Nuclear extracts of untransfected or p300-siRNA-transfected CLL cells from 2 patients were incubated with biotinylated DNA harboring STAT3 binding sites. EMSA showed that the addition of excess unlabeled probe, anti-STAT3 antibodies, but not their isotype IgG, or transfection with p300-siRNA, but not with GAPDH, attenuated the binding of the cell extract to the labeled DNA probe, suggesting that transfection with p300-siRNA inhibits STAT3-DNA binding. ( B ) CLL cells were transfected with p300-siRNA or with GAPDH and qRT-PCR was used to determine the levels of STAT3-regulated genes. As shown, levels of CASP3, c-Myc, P21, VEGF, and STAT3 mRNA levels were downregulated in p300-siRNA-transfected cells. ( C ) Flow cytometry analysis of CLL cells transfected with p300-siRNA or with GAPDH. Compared with GAPDH-transfected cells the rate of active apoptosis (Annexin/PI positive) were 3 folds higher in p300-siRNA transfected cells.

Article Snippet: After blocking, the membrane was incubated with the following primary antibodies: monoclonal mouse anti-human STAT3 (BD Bioscience, Palo Alto, CA, USA), monoclonal mouse anti-human Ser 727 STAT3 (Cell Signaling, Danvers, MA, USA), Rabbit anti-human acetyl STAT3 (Lys 685) antibodies (Cell Signaling), mouse anti-human p300 antibodies (Pierce Biotechnology Inc./Thermo scientific, Rockville, IL), and mouse anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Incubation, Binding Assay, Labeling, Quantitative RT-PCR, Flow Cytometry

Journal: Cancer research

Article Title: An ARC-regulated IL1β/Cox-2/PGE2/β-catenin/ARC circuit controls leukemia-microenvironment interactions and confers drug resistance in AML

doi: 10.1158/0008-5472.CAN-18-0921

Figure Lengend Snippet: AML cells treated dmPGE2 or co-cultured with MSCs express increased β-catenin and ARC and are more resistance to chemotherapy. A. OCI-AML3 cells were treated with dmPGE2 (1 or 4 μM) or co-cultured with MSCs without or with Cox-2 inhibitor Celecoxib (200 nM) for 48 h. β-catenin and ARC protein levels were determined by western blot. For co-culture experiments, OCI-AML3 cells were FACS-sorted after co-culture and west blot was done using the lysate from the sorted CD45+CD90− cells as shown in the histogram. B. OCI-AML3 cells were treated with dmPGE2 (4 μM) or co-cultured with MSCs for 48 h. Cytosolic and nuclear β-catenin levels were determined by western blot. C. OCI-AML cells were treated with Ara-C with or without dmPGE2 (1 or 4 μM) or with MSC co-culture in the absence or presence of Cox-2 inhibitor Celecoxib (200 nM) for 48 and 72 h. Apoptosis was assessed by flow cytometry. COX, co-culture.

Article Snippet: For ARC detection, tissues were stained overnight at 4°C with an anti-ARC antibody (1:4000, Cat#38916S, Cell Signaling Technology) and for β-catenin detection, they were incubated 1 h at room temperature with an anti-β-catenin antibody (1:1000; Cat#LS-B4123, LifeSpan BioSciences Inc.; Seattle, WA).

Techniques: Cell Culture, Western Blot, Co-Culture Assay, Flow Cytometry

Journal: Cancer research

Article Title: An ARC-regulated IL1β/Cox-2/PGE2/β-catenin/ARC circuit controls leukemia-microenvironment interactions and confers drug resistance in AML

doi: 10.1158/0008-5472.CAN-18-0921

Figure Lengend Snippet: β-catenin regulates ARC expression in AML cells. A. OCI-AML3 cells were transfected with a control siRNA (scramble) or smart pool siRNAs against β-catenin gene (CTNNB1 siRNA) (750 nM) by Amaxa electroporation. The RNA (24 h) and protein (24 and 48 h) levels of β-catenin and ARC were determined by RT-PCR and western blot, respectively, after transfection. CON, scramble control. MWM, molecular weight marker. B. OCI-AML3 cells transduced with ARC promoter-driven GFP reporter gene were transfected with scramble control or β-catenin siRNAs and GFP levels were determined 24 h after transfection. C. OCI-AML3 cells transduced with ARC promoter (WT)- or ARC mutant promoter-driven GFP reporter gene were treated with PGE2 or C-82 for 48 h. For B and C, histograms (top panels) are representative results from one experiment and bar grafts (lower panels) are results of triplicates. GFP levels were determined by flow cytometry. Untransduced OCI-AML3 cells were used as a negative control for GFP.

Article Snippet: For ARC detection, tissues were stained overnight at 4°C with an anti-ARC antibody (1:4000, Cat#38916S, Cell Signaling Technology) and for β-catenin detection, they were incubated 1 h at room temperature with an anti-β-catenin antibody (1:1000; Cat#LS-B4123, LifeSpan BioSciences Inc.; Seattle, WA).

Techniques: Expressing, Transfection, Electroporation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Molecular Weight, Marker, Transduction, Mutagenesis, Flow Cytometry, Negative Control

Journal: Cancer research

Article Title: An ARC-regulated IL1β/Cox-2/PGE2/β-catenin/ARC circuit controls leukemia-microenvironment interactions and confers drug resistance in AML

doi: 10.1158/0008-5472.CAN-18-0921

Figure Lengend Snippet: Inhibition of ARC in AML cells reduces serum IL1β and PGE2 levels, decreases leukemia burden in various tissues, prolongs survivals, and sensitizes to Ara-C in NSGS mice. A. In vivo experiment scheme. B. IL1β and PGE2 levels in serum of mice harboring control or ARC KD OCI-AML3 cells determined by ELISA. C. huCD45 positivity in BM and spleen of mice harboring control or ARC KD OCI-AML3 cells determined by flow cytometry. D. Liver and spleen from mice harboring control or ARC KD OCI-AML3 cells. E. Expression of ARC and β-catenin in spleen huCD45+ cells of mice harboring control or ARC KD OCI-AML3 cells with or without Ara-C treatment, determined by Opal/TSA multiplex IHC staining and Vectra multispectral imaging analysis. Left, huCD45 (yellow), ARC (red), β-catenin (cyan), DAPI (blue), and merged imaging (objective lens ×20, scale bar 50 µm). Fluorophores for CD45, ARC, and β-catenin are Opal 540, Opal 570 and Opal 650, respectively. Right, the quantitation of ARC and β-catenin expression in huCD45+ cells of mouse spleen: 35 fields in control cell-injected mice (7,164 to 10,873 cells/field, total 319,431 cells), 26 fields in control cell-injected mice with Ara-C treatment (2,635 to 4,777 cells/field, total 99,727 cells), 17 fields in ARC KD cell-injected mice (610 to 5,021 cells/fields, total 37,206 cells), and 22 fields in ARC KD cell-injected mice with Ara-C treatment (248 to 3,927 cells/field, total 45,431 cells); respectively. F. huCD45 positivity in PB of mice harboring control or ARC KD OCI-AML3 cells with or without Ara-C treatment by flow cytometry. G. Survival of mice injected with control or ARC KD OCI-AML3 cells without or with Ara-C treatment.

Article Snippet: For ARC detection, tissues were stained overnight at 4°C with an anti-ARC antibody (1:4000, Cat#38916S, Cell Signaling Technology) and for β-catenin detection, they were incubated 1 h at room temperature with an anti-β-catenin antibody (1:1000; Cat#LS-B4123, LifeSpan BioSciences Inc.; Seattle, WA).

Techniques: Inhibition, In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Multiplex Assay, Immunohistochemistry, Imaging, Quantitation Assay, Injection

Journal: Cancer research

Article Title: An ARC-regulated IL1β/Cox-2/PGE2/β-catenin/ARC circuit controls leukemia-microenvironment interactions and confers drug resistance in AML

doi: 10.1158/0008-5472.CAN-18-0921

Figure Lengend Snippet: PGE2/β-catenin/ARC cascade and targeting in primary AML samples and the proposed mechanism of ARC action. A. IL1β and PGE2 levels in BM samples from AML patients (n = 8) (Table 1, samples 9-16) and normal controls (NBM, n = 4) by ELISA. B. Correlation of ARC and β-catenin protein levels, determined by CyTOF in various BM cell populations of AML patients (n = 4) (Table 1, samples 17-20). C. Levels of ARC protein, determined by CyTOF in AML patient samples (n = 2) (Table 1, samples 21 and 22) treated with β-catenin inhibitor C-82 (0.5 μM) without or with MSC co-culture for 48 h. Protein levels determined by CyTOF are expressed as Arcsinh-transformed counts. D. AML patient samples were treated with C-82, Ara-C, or both for 48 h. Apoptosis was determined in blasts (n = 5) (Table 1, samples 23-27) and CD34+CD38− (n = 4) (Table 1, samples 23-26) cells. cocx, co-culture. E. Proposed mechanism of action: ARC, regulated by β-catenin, mediates leukemia stromal interaction through ARC-IL1β/Cox-2/PGE2/β-catenin circuit.

Article Snippet: For ARC detection, tissues were stained overnight at 4°C with an anti-ARC antibody (1:4000, Cat#38916S, Cell Signaling Technology) and for β-catenin detection, they were incubated 1 h at room temperature with an anti-β-catenin antibody (1:1000; Cat#LS-B4123, LifeSpan BioSciences Inc.; Seattle, WA).

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transformation Assay

Journal: eLife

Article Title: Binary outcomes of enhancer activity underlie stable random monoallelic expression

doi: 10.7554/eLife.74204

Figure Lengend Snippet:

Article Snippet: Cell Signaling: anti-H3K27me3 (C36B11), anti-H2AUb1 (D27C4).

Techniques: Generated, Microinjection, Electroporation, CRISPR, Purification, cDNA Synthesis, Cell Isolation, Multiplex Assay, TA Cloning, Subcloning, Staining, Flow Cytometry, Recombinant, Cell Culture, Software

Journal: Radiation Oncology (London, England)

Article Title: Regulation of protein translation initiation in response to ionizing radiation

doi: 10.1186/1748-717X-8-35

Figure Lengend Snippet: IR induced caspase-dependent processing of eIF4G1, eIF4B, as well as eIF3A, and caused disassembly of the cap-dependent initiation complex. Jurkat cells were irradiated with 10 Gy. ( A ) Lysates were made 4–24 h after IR. Cleavage of eIF4G1 and eIF3A, as well as eIF4B decline, were observed in response to IR. The cleavage coincided with processing of caspase-3 (C3) and the caspase-3 substrate PARP indicating caspase-3 activation. No IR-induced processing of eIF4E, eIF4A, and eIF4H was detected. Densitometric analysis shows relative protein levels normalized to the respective levels in non-irradiated cells (0h after IR). ( B ) 24 h after irradiation, cells were lysed and a pull-down assay was made using 7-methyl GTP agarose to mimic the cap structure of mRNAs. Whereas the recruitment of eIF4E to the cap structure remained unchanged, recruitment of eIF4A, eIF4G1, and eIF3A into the cap-dependent initiation complex was reduced after IR. ( C ) Jurkat cells were irradiated with 10 Gy and co-treated with 30 μM of caspase inhbitor zVAD or the respective amount of solvent. 24 h later, cells were lysed. Treatment with zVAD blocked IR-induced cleavage of eIF4G1, eIF3A, PARP, and the decline of eIF4B, as well as the processing of caspase-3 to the active p19 and p17 forms indicating that the processing of the three initiation factor occurred after caspase activation. However, caspase inhibitor zVAD did not block IR-induced 4EBP1 dephsophorylation on Mcl-1 reduction.

Article Snippet: Following antibodies were used for Western blotting and immunoprecipitation: mouse-anti β-actin from Sigma (Deisenhofen, Germany), mouse-anti GAPDH from Abcam (Cambridge, UK), rabbit-anti Akt, phospho-Akt (S473), phospho-Akt (T308), mTOR, phospho-mTOR (S2448), phospho-mTOR (S2481), S6K, phospho-S6K (T389), 4EBP1, phospho-4EBP1 (T37/46), phospho-4EBP1 (T70), Mcl-1, eIF3A, eIF4A, eIF4B, eIF4E, eIF4G, phospho-eIF4G, and DAP5 from Cell Signaling (NEB, Frankfurt, Germany).

Techniques: Irradiation, Activation Assay, Pull Down Assay, Solvent, Blocking Assay

Journal: Radiation Oncology (London, England)

Article Title: Regulation of protein translation initiation in response to ionizing radiation

doi: 10.1186/1748-717X-8-35

Figure Lengend Snippet: IR induced 4EBP1 dephoshorylation independent of Akt/mTOR. Jurkat cells were treated with 50 μM or 100 μM LY294002 (LY) ( A ) or irradiated with 10 Gy ( B ). Lysates were made 6 h after treatment with LY294002 or 4 h, 8 h, 12 h, and 24 h after IR. ( A ) LY294002 induced dephosphorylation of Akt on threonine 308 (T308) and serine 473 (S473) and on mTOR on serine 2448 and 2481 (S2448, S2481) indicating an inhibition of the Akt/mTOR pathway. Dephosphorylation of p70S6K on threonine 389 (T389) of p85S6K on threonine 412 (T412), and of 4EBP1 on threonine 37/46 (T37/46) and threonine 70 (T70) was also observed after treatment with LY294002. ( B ) IR did not affect phospho-Akt, phospho-mTOR, and phospho-S6K levels, but reduced phospho-4EBP1 levels as indicated by anti-phospho-4EBP1 antibodies and a shift of 4EBP1 from p20 to p17, suggesting that IR induced 4EBP1 dephoshphorylation independent of Akt/mTOR pathway. Reduction of Mcl-1 levels in response to LY294002 and IR correlated with 4EBP1 dephosphorylation. ( C, D ) Jurkat cells were treated with 50 μM or 100 μM LY294002. 6 h and 24 h later, mitochondrial dissipation (ΔΨm low, C ) and DNA fragmentation (sub G1, D ) were analyzed by flow cytometry. LY294002 induced mitochondrial dissipation and DNA fragmentation in a time- and concentration-dependent manner. (E) Jurkat cells were irradiated with 10 Gy. 4–24 h after irradiation, cells were lysed. Phospho-ERK1/2 and ERK levels were analyzed by western blot. No changes of phospho-ERK1/2 and ERK1/2 levels were observed in response to IR. Flow cytometric data shows mean values ± S.D. (n = 3), ** indicates p < 0.01, *** indicates p < 0.001, n.s. indicates no significance (p > 0.05).

Article Snippet: Following antibodies were used for Western blotting and immunoprecipitation: mouse-anti β-actin from Sigma (Deisenhofen, Germany), mouse-anti GAPDH from Abcam (Cambridge, UK), rabbit-anti Akt, phospho-Akt (S473), phospho-Akt (T308), mTOR, phospho-mTOR (S2448), phospho-mTOR (S2481), S6K, phospho-S6K (T389), 4EBP1, phospho-4EBP1 (T37/46), phospho-4EBP1 (T70), Mcl-1, eIF3A, eIF4A, eIF4B, eIF4E, eIF4G, phospho-eIF4G, and DAP5 from Cell Signaling (NEB, Frankfurt, Germany).

Techniques: Irradiation, De-Phosphorylation Assay, Inhibition, Flow Cytometry, Concentration Assay, Western Blot

Journal: Radiation Oncology (London, England)

Article Title: Regulation of protein translation initiation in response to ionizing radiation

doi: 10.1186/1748-717X-8-35

Figure Lengend Snippet: Radiation-induced Mcl-1 decline and apoptosis was not affected by silencing of 4EBP1. ( A, B ) Jurkat cells were irradiated with 10 Gy. ( A ) 24 h after IR cells were lysed and immunoprecipitation was performed using antibodies against 4EBP1 or eIF4E. As negative controls, isotype-matched antibodies (IgG) were used. Equal input was verified by western blot. Interaction of 4EBP1 with eIF4E was enhanced in irradiated cells. ( B ) 24 h after irradiation, cells were lysed and and a pull-down assay was made using 7-methyl GTP agarose. 4EBP1 with eIF4E bound to 7-methyl GTP cap. ( C-E ) Jurkat cells were transfected with 1 μM 4ebp1 siRNA or non-targeting (nt) siRNA by electroporation. 48 h later, cells were irradiated with 10 Gy. ( C ) 24 h after irradiation, cells were lysed. 4EBP1 and Mcl-1 protein levels were analyzed by densitometry and normalized to the respective levels in non-irradiated cells transfected with non-targeting siRNA. Silencing of 4EBP1 had hardly any effect on Mcl-1 protein levels. Dissipation of the mitochondrial membrane potential (ΔΨm low, D ) and DNA fragmentation (sub G1, E ) were analyzed by flow cytometry 24 h and 48 h after IR, respectively. ( F ) Jurkat cells were transfected with 250 nM mcl1 siRNA. Lysates were made 3 h and 6 h after electroporation. Mcl-1 protein levels were analyzed by densitometry and normalized to the Mcl-1 level in lysates made 3 h after transfection with non-targeting siRNA. Down-regulation of Mcl-1 by siRNA resulted in a rapid dephosphorylation of 4EBP1 which was indicated by the shift to faster migrating p17 band. Flow cytometric data show mean values ± S.D. (n = 3), n.s. indicates no significance (p > 0.05).

Article Snippet: Following antibodies were used for Western blotting and immunoprecipitation: mouse-anti β-actin from Sigma (Deisenhofen, Germany), mouse-anti GAPDH from Abcam (Cambridge, UK), rabbit-anti Akt, phospho-Akt (S473), phospho-Akt (T308), mTOR, phospho-mTOR (S2448), phospho-mTOR (S2481), S6K, phospho-S6K (T389), 4EBP1, phospho-4EBP1 (T37/46), phospho-4EBP1 (T70), Mcl-1, eIF3A, eIF4A, eIF4B, eIF4E, eIF4G, phospho-eIF4G, and DAP5 from Cell Signaling (NEB, Frankfurt, Germany).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Pull Down Assay, Transfection, Electroporation, Membrane, Flow Cytometry, De-Phosphorylation Assay

Journal: Radiation Oncology (London, England)

Article Title: Regulation of protein translation initiation in response to ionizing radiation

doi: 10.1186/1748-717X-8-35

Figure Lengend Snippet: Regulation of proteins translation in Jurkat cells. Schema shows signaling pathway regulating cap-dependent translation in non-irradiated (left) and irradiated (right) cells. In non-irradiated Jurkat cells, the Akt signaling pathway is constitutively activated. Active Akt, indicated by two phosphorylation sites, phosphorylates and activates the kinase mTOR which in turn upregulates the ribosomal translation through phosphorylation of S6K. In addition, phosphorylation of 4EBP1 prevents the protein from binding to the initiation factor eIF4E and allows eIF4E to recruit eIF4G, eIF4A and the eIF3 complex containing the eIF3A subunit to the cap structure at the 5’ end of the mRNA. Thus assembled, the complex is able to initiate protein translation. eIF4B supports eIF4A thereby positively regulating protein translation. IR had no effect on Akt activity. Therefore, Akt and mTOR as well as S6K remain phosphorylated and activated 24 h after irradiation. In contrast, 4EBP1 is dephosphorylated in response to IR, probably due to an enhanced phosphatase activity. Hypophosphorylated 4EBP1 associates with eIF4E and prevents the recruitment of eIF4G to the cap structure. Furthermore, eIF4G, eIF3A, and eIF4B are cleaved downstream of caspase activation (indicated by the white line). As a consequence, the pre-initiation complex is disassembled and cap-dependent translation averted. In addition to eIF4G, eIF4A and the eIF3A complex associate with DAP5 in non-irradiated Jurkat cells suggesting a cap-independent protein translation through an alternative initiation site. In response to IR, DAP5 is cleaved in a caspase-dependent manner. The cleavage coincides with a disassembly of the DAP5-dependent initiation complex, probably resulting in reduced DAP5-dependent translation.

Article Snippet: Following antibodies were used for Western blotting and immunoprecipitation: mouse-anti β-actin from Sigma (Deisenhofen, Germany), mouse-anti GAPDH from Abcam (Cambridge, UK), rabbit-anti Akt, phospho-Akt (S473), phospho-Akt (T308), mTOR, phospho-mTOR (S2448), phospho-mTOR (S2481), S6K, phospho-S6K (T389), 4EBP1, phospho-4EBP1 (T37/46), phospho-4EBP1 (T70), Mcl-1, eIF3A, eIF4A, eIF4B, eIF4E, eIF4G, phospho-eIF4G, and DAP5 from Cell Signaling (NEB, Frankfurt, Germany).

Techniques: Irradiation, Phospho-proteomics, Protein Binding, Activity Assay, Activation Assay

Journal: Oncotarget

Article Title: YM155 exerts potent cytotoxic activity against quiescent (G 0 /G 1 ) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1

doi: 10.18632/oncotarget.22871

Figure Lengend Snippet: (A) The cells (KMS12, KMS11, and U266) were treated with 0.1 μM YM155 for indicated time periods (control: 0.1% DMSO). (B) The cells were treated with 10, 100, or 300 nM YM155 for 24 hr. Expression levels of survivin and Mcl-1 protein were analyzed by immunoblotting (left panel) as described in Materials and Methods. Histograms (right panels) represent the ratio of band intensity of drug-treated to vehicle-treated, each normalized to background. Error bars represent SD from triplicate experiments. (C) Expression levels of survivin and Mcl-1 mRNA were analyzed by real-time RT-PCR analysis using 18s rRNA as an internal control. The cells (KMS12, KMS11, and U266) were treated with 1 μM YM155 for indicated time periods (control: 0.1% DMSO). After drug treatment, total RNA was prepared. Colums, mean from three separate experiments. * P<0.05, ** P<0.01. (D) The U266 cells were incubated with 0.1 μM YM155 in the presence or absence of 30 μM Z-VAD-FMK for 24 hr. Z-VAD-FMK was added 60 min before addition of YM155. Survivin and Mcl-1 protein expression was analyzed by immunoblotting (left panel). Histograms (right panels) represent the ratio of band intensity of drug-treated to vehicle-treated, each normalized to background. Error bars represent SD from triplicate experiments. (E) The U266 cells were incubated with 0.1 μM YM155 or 20μM MG-132, alone or in combinations for 6 hours. MG-132 was added 60 min before addition of YM155. Mcl-1 protein expression was analyzed by immunoblotting (left panel). Histograms (right panels) represent the ratio of band intensity of drug-treated to vehicle-treated, each normalized to background. Error bars represent SD from triplicate experiments. ** P<0.01.

Article Snippet: The following antibodies were used: Survivin, STAT3, Mcl-1, Bcl-xL and c-Myc from Cell Signaling Technology (Danvers, MA); phospho- STAT3 from American Research Products (Waltham, MA); Actin from Sigma-Aldrich.

Techniques: Control, Expressing, Western Blot, Quantitative RT-PCR, Incubation

Journal: Oncotarget

Article Title: YM155 exerts potent cytotoxic activity against quiescent (G 0 /G 1 ) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1

doi: 10.18632/oncotarget.22871

Figure Lengend Snippet: U266 cells were transduced with survivin-GFP or Mcl-1 retrovirus constructs. Ectopic expressions of survivin or Mcl-1 were confirmed by immunoblotting (right panels). The cells were incubated with various concentrations of YM155 at 37°c for 72 h. Cell growth inhibition rate was determined by trypan blue dye exclusion assay. All quantitative data are shown as the mean of three independent experiments. Error bars represent SD from triplicate experiments.

Article Snippet: The following antibodies were used: Survivin, STAT3, Mcl-1, Bcl-xL and c-Myc from Cell Signaling Technology (Danvers, MA); phospho- STAT3 from American Research Products (Waltham, MA); Actin from Sigma-Aldrich.

Techniques: Transduction, Construct, Western Blot, Incubation, Inhibition, Exclusion Assay

Journal: Oncotarget

Article Title: YM155 exerts potent cytotoxic activity against quiescent (G 0 /G 1 ) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1

doi: 10.18632/oncotarget.22871

Figure Lengend Snippet: (A) KMS12 cells were incubated with or without IL-6 (100 ng/ml) for indicated time periods. The cell growth was determined by trypan blue dye exclusion assay. All quantitative data are shown as the mean of three independent experiments. Error bars represent SD from triplicate experiments. (B) KMS12 cells were incubated with IL-6 (100 ng/ml) for 9 h in the presence or absence of YM155. YM155 was added 60 min after addition of IL-6. The phosphorylation of STAT3 and the expression of Mcl-1 and survivin protein were analyzed by immunoblotting analysis as described in Materials and Methods. (C) The expression levels of Bcl-xl and c-Myc protein were analyzed by immunoblotting. KMS11 cells were treated with 0.1 μM YM155 for indicated time periods (control: 0.1% DMSO). (D) KMS12 cells were incubated with YM155 at indicated concentrations in the absence or presence of IL-6 (100 ng/ml) for 48 hr. YM155 was added 60 min after addition of IL-6. The cell viability was determined by trypan blue dye exclusion assay. All quantitative data are shown as the mean of three independent experiments. Error bars represent SD from triplicate experiments.

Article Snippet: The following antibodies were used: Survivin, STAT3, Mcl-1, Bcl-xL and c-Myc from Cell Signaling Technology (Danvers, MA); phospho- STAT3 from American Research Products (Waltham, MA); Actin from Sigma-Aldrich.

Techniques: Incubation, Exclusion Assay, Phospho-proteomics, Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: YM155 exerts potent cytotoxic activity against quiescent (G 0 /G 1 ) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1

doi: 10.18632/oncotarget.22871

Figure Lengend Snippet: (A) U266 cells were incubated in 0.1% or 10% FBS medium for 72 h, and cell-cycle profiles were examined by flow cytometry. (B) U266 cells were cultured in 0.1% or 10% FBS medium for 72 h, then the cells were incubated with various concentrations of YM155, 0.1 mM ara-C or 3 nM bortezomib at 37°c for indicated time periods. Cell survival inhibition rate was determined by trypan blue dye exclusion assay. (C) U266 cells were cultured in 0.1% or 10% FBS medium for 72h. RPMI8226 cells were cultured in 0.2% or 10% FBS medium for 48h. Flow cytometry was done to investigate G 0 populations by double staining DNA with Hoechst33342 (Hst) and RNA with pyronin Y (PY). The G 0 population (2N DNA/low levels of RNA, Hst+/PY-) was discriminated from the G 1 population (2N DNA/high levels of RNA, Hst+/PY+). Results are representative of 3 separate sets of experiments. (D) G 0 -riched U266 or RPMI8226 cells were cultured in presence or absence (control: 0.1% DMSO) of YM155 (1 μM or 100 nM) for 24 hr. Triple color flow cytometeric analysis was performed to assess cell death (7-AAD positive cells) in G 0 and G 1 populations compared with control. Results (7-AAD positive cells) are representative of 3 separate sets of experiments. (E) U266 cells were cultured in low-serum-containing medium for 72 h, then the cells were incubated with 0.1 μM YM155 at 37°c for indicated time periods. The expression of survivin and Mcl-1 protein was analyzed by immunoblotting analysis. Histograms (right panels) represent the ratio of band intensity of drug-treated to vehicle-treated, each normalized to background.

Article Snippet: The following antibodies were used: Survivin, STAT3, Mcl-1, Bcl-xL and c-Myc from Cell Signaling Technology (Danvers, MA); phospho- STAT3 from American Research Products (Waltham, MA); Actin from Sigma-Aldrich.

Techniques: Incubation, Flow Cytometry, Cell Culture, Inhibition, Exclusion Assay, Double Staining, Control, Expressing, Western Blot

Journal: Oncotarget

Article Title: YM155 exerts potent cytotoxic activity against quiescent (G 0 /G 1 ) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1

doi: 10.18632/oncotarget.22871

Figure Lengend Snippet: (A) Cell growth inhibition by bortezomib in U266 or U266/BTZR1 cells. (B) Cell growth inhibition by YM155 in U266 or U266/BTZR1 cells. The cells were incubated with various concentrations of bortezomib (A) or YM155 (B) at 37°c for 72 h. Cell growth inhibition rate was determined by Cell counting Kit as described in Materials and Methods. IC 50 was calculated as the mean of three independent experiments with triplicate determinations at each concentration. (C) Expression levels of survivin and Mcl-1 protein in U266 or U266/BTZR1 cells were analyzed by immunoblotting (left panel). Histograms (right panels) represent the ratio of band intensity compared to its parental cells, each normalized to background and band intensity of actin. Error bars represent standard deviation (SD, n ≥ 3). (D) U266/BTZR1 cells were transfected with siRNA against survivin or Mcl-1 by electroporation. Cells were also transfected with control siRNA. Proteins were extracted from the transfected cells 24 h after electroporation. Expression levels of survivin and Mcl-1 protein were analyzed by immunoblotting as described in Materials and Methods (left panel). Twenty-four hours after electroporation, U266 cells were incubated with 30 nM bortezomib for 72 h. The cell growth was determined by trypan blue dye exclusion assay. All quantitative data are shown as the mean of three independent experiments. Error bars represent SD from triplicate experiments (right panel). (E) Expression levels of survivin and Mcl-1 protein were analyzed in U266/BTZR1 cells incubated with YM155 by immunoblotting (left panel). The U266/BTZR1 cells were treated with 0.1 μM YM155 for indicated time periods (control: 0.1% DMSO). Histograms (right panels) represent the ratio of band intensity of drug-treated to vehicle-treated, each normalized to background. Error bars represent standard deviation (SD, n ≥ 3).

Article Snippet: The following antibodies were used: Survivin, STAT3, Mcl-1, Bcl-xL and c-Myc from Cell Signaling Technology (Danvers, MA); phospho- STAT3 from American Research Products (Waltham, MA); Actin from Sigma-Aldrich.

Techniques: Inhibition, Incubation, Cell Counting, Concentration Assay, Expressing, Western Blot, Standard Deviation, Transfection, Electroporation, Control, Exclusion Assay