Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Gammaretrovirus-based Gag.MS2 chimera for CRISPR-Cas9 delivery (A) Design of the gammaretroviral Gag.MS2 (g.Gag.MS2) variant and non-retroviral SpCas9.TS and sgRNA.TS expression plasmids. The configuration of structural Gag and enzymatic Pol proteins within gammaretroviral wt Gag-Pol (wt g.Gag-Pol) is depicted at the top. It consists of matrix (MA), p12, capsid (CA), nucleocapsid (NC), protease (PR), reverse transcriptase (RT), and integrase (IN) subdomains, which are separated from each other by individual protease sites. In g.Gag.MS2, the MS2CP dimer (2× MS2CP) protein replaces NC while maintaining the natural retroviral protease site (black bold bar). To ensure specific packaging of CRISPR-Cas9 RNA into assembling g.Gag.MS2 particles, two copies of MS2 target site (TS) are incorporated downstream of EGFP within the SpCas9.TS expression plasmid or were placed in (TS.inc) or adjacent (TS.adj) to the sgRNA scaffold in respective sgRNA expression plasmids. Co-expression of EGFP and DsRedexp helped to monitor transfection during particle production. (B) Direct comparison of integrating (g.RIT) and non-integrating g.Gag.MS2 CRISPR.Tet2 particles. Indicated supernatant volumes were used to transduce human HT1080-based RFP657.Tet2 reporter cells. Each data point represents one individually generated supernatant (n = 3). CMV, promoter from cytomegalovirus; hU6, human RNA Pol III U6 promoter; pA, poly(A) signal; PRE, post-transcriptional regulatory element from woodchuck hepatitis virus; SFFV, promoter from spleen focus forming virus.

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Variant Assay, Retroviral, Expressing, Reverse Transcription, Plasmid Preparation, Transfection, Comparison, Transduction, Generated, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Alpharetrovirus-based Gag.MS2 particles showed improved delivery of CRISPR-Cas9 components into target cells (A) Design of alpharetroviral Gag.MS2 (a.Gag.MS2) variants. The wt alpharetroviral Gag-Pol polyprotein (wt a.Gag-Pol) is shown at the top of the figure panel. In contrast to g.Gag-Pol, alpharetroviral PR is part of the Gag open reading frame (ORF) (and not Pol ORF). In the depicted a.Gag.MS2 variant, the MS2CP dimer was separated from NC by the naturally occuring viral protease site (black bold bar). (B) a.Gag.MS2.CRISPR.Tet2 particles containing the Tet2.TS.inc sgRNA variant depict higher knockout rates compared with their Tet2.TS.adj counterparts. Indicated volumes of supernatants were used to transduce HT1080-based RFP657.Tet2 reporter cells. The graph depicts RFP657.Tet2 knockout rates mediated by three independent supernatants (n = 3). (C) a.Gag.MS2.CRISPR.Tet2 particles are superior to their gammaretroviral counterparts. Direct comparison of three individually produced a.Gag.MS2- and g.Gag.MS2-based CRISPR.Tet2 supernatants in HT1080-based RFP657.Tet2 reporter cells is shown (n = 3). (D) a.Gag.MS2.CRISPR.Tet2 particles mediate efficient targeted gene knockout in hiPSCs. hiPSC-based RFP657.Tet2 reporter cells were transduced with indicated volumes of three different batches of g.Gag.MS2.CRISPR.Tet2 or a.Gag.MS2.CRISPR.Tet2 supernatants (n = 3). A LIT.CRISPR.Tet2 supernatant applied at MOI 2 (2.9 μL) or MOI 5 (7.4 μL) served as a positive control (n = 1).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Variant Assay, Knock-Out, Transduction, Comparison, Produced, Gene Knockout, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Characterization of alpha- and gammaretroviral Gag.MS2.CRISPR.Tet2 supernatants In total, two different batches (white and black filled circles), each with three individually packaged supernatants for a.Gag.MS2 and g.Gag.MS2 CRISPR.Tet2 particles, were generated and characterized for their content of CRISPR-Cas9 components (n = 6). (A) SpCas9.TS mRNA and sgRNA.TS content in a.Gag.MS2- and g.Gag.MS2-based CRISPR.Tet2 supernatants. Total RNA copies per microliter supernatant were calculated using individual plasmid standards. (B) Gag.MS2.CRISPR.Tet2 supernatants contain SpCas9 protein. SpCas9 protein concentrations were assessed by an SpCas9 ELISA. (C) a.Gag.MS2.CRISPR.Tet2 supernatants had a higher Gag.MS2 precursor protein content. Immunoblot analysis of three individually packaged a.Gag.MS2.CRISPR.Tet2 and g.Gag.MS2.CRISPR.Tet2 supernatants is shown (batch 2; black filled circles) (n = 3). g.Gag.MS2 (82 kDa) and a.Gag.MS2 (89 kDa) precursor proteins were detected with an anti-MS2 antibody. Ponceau S staining of the membrane is shown below. (D) g.Gag.MS2.CRISPR.Tet2-mediated RFP657.Tet2 knockout rates can be adjusted by ∼10-fold higher supernatant volumes. Characterized supernatants from batch 2 were used to transduce HT1080 RFP657.Tet2 reporter cells at the depicted volumes (n = 3). (E) Normalization of supernatant volumes showed an ∼2-fold-higher potency for a.Gag.MS2-based CRISPR.Tet2 particles. The in (D) acquired datasets were replotted as % RFP657.Tet2 knockout against applied SpCas9.TS mRNA copies/cell (n = 3; mean values are depicted).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Generated, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Membrane, Knock-Out, Transduction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Efficient CRISPR-mediated transition of EGFP to EBFP by a.Gag.MS2.CRISPR.HDR particles (A) Determined copies of CRISPR RNA and PT-modified ssODN molecules within a.Gag.MS2.CRISPR.HDR supernatants. (B and C) HT1080-based (B) or NuFF-based (C) EGFP reporter cells were transduced with 30 μL (1,980 copies SpCas9.TS mRNA/cell) of a.Gag.MS2.CRISPR.HDR supernatants in the absence (w/o) or presence of NHEJ or HDR inhibitors (inh) M3814 or YU238259, respectively. DMSO-treated cells served as solvent controls. The graphs display % EBFP + cells within treated cultures. Non-treated or DMSO-treated mock cultures indicate the degree of autofluorescence in the EBFP channel. Each data point reflects one independently prepared supernatant (n = 3–4).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Modification, Transduction, Solvent

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress oxidative stress–induced parthanatos. A , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: MTS Assay, Control, Concentration Assay, Labeling, Western Blot, Software, Fluorescence, FACS

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress the ALIS formation. A , HT1080 and p62 KO HT1080 cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. B , HT1080 cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. C , HT1080 cells were treated with CTX (1 mg/ml) and/or NAC (2 mM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. D , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. Scale bar represents 10 μm. G , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. I , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were treated with the indicated reagents for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. CTX (1 mg/ml). I3MT-3 (10 μM). PAG (5 mM). Scale bar represents 10 μm. K , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, I3MT-3 0 μM PAG 0 mM cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; PAG, DL-propargylglycine; NAC, N-acetylcysteine; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS disaggregate the ALIS by inducing HSP70. A , HT1080 cells were treated with the indicated reagents for 24 h and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. CTX (1 mg/ml). PAG (5 mM). I3MT-3 (20 μM). Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM, I3MT-3 0 μM cells). B , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h and then incubated with 10 μM DCFH-DA. Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), N.S. p > 0.05 ( versus CTX 1 mg/ml Na 2 S 4 0 μM cells). C , HT1080 cells were treated with the indicated reagents for 24 h, and then the detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. Bafilomycin A1 (5 nM). CTX (1 mg/ml). Na 2 S 4 (100 μM). D , HT1080 cells were treated with CTX (1 mg/ml) for 20 h and then treated with MG132 (10 μM) and/or Na 2 S 4 (100 μM) for 4 h. The detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the indicated concentration of Na 2 S 4 for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. G , HT1080 cells were treated with CTX (1 mg/ml) and Na 2 S 4 (100 μM) for 18 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then whole cell lysate were subjected to immunoblotting with the indicated antibodies. I , HT1080 and PARP-1 KO cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. L , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05, ∗∗ p < 0.01, ( versus siRNA Ctr, CTX 1 mg/ml cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; HSP, heat shock protein; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; ROS, reactive oxygen species; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Incubation, Fluorescence, Control, Western Blot, Concentration Assay, Transfection, Negative Control, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS activate HSF1 by promoting its dissociation from HSP90. A , HT1080 cells were treated with the Na 2 S 4 (100 μM) for indicated period, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗∗ p < 0.001, ( versus control cells). B , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. C , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. D , HT1080 and PARP-1 KO cells were treated with the Na 2 S 4 (100 μM) for 6 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus control cells), ## p < 0.01 ( versus Na 2 S 4 100 μM, KRIBB11 0 μM cells). G , HT1080 cells were transfected with FLAG-HSP90 and/or Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with indicated concentration of Na 2 S 4 for 4 h, then immunoprecipitated protein G-Sepharose beads with the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. I , HSP90 was immunoprecipitated using anti-HSP90 antibody with protein G beads. Beads were washed four times with PBS and then treated with Na 2 S 4 (100, 1000 μM) for 1 h. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with FLAG-HSP90 (WT/C412A/C564A/C521A) and Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with FLAG-Empty or FLAG-HSP90 (WT/C521A) plasmid for 24 h and treated with Na 2 S 4 (100 μM) for indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. All data are representative of at least three independent experiments. HSF, heat shock factor; HSP, heat shock protein; PARP-1, poly (ADP-ribose) polymerase-1; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Concentration Assay

Journal: Cell Death & Disease

Article Title: Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells

doi: 10.1038/cddis.2014.497

Figure Lengend Snippet: Effect of different concentrations of androgens on DNA synthesis of mesenchymal cells. Quiescent NIH3T3 cells were used and left untreated or treated with the indicated compounds. R1881 (Perkin-Elmer) or DHT (Sigma) were used at 1 pM or 10 nM; bicalutamide (Sigma-Aldrich; Bic) was added at 1000-fold excess. In ( a and c ), cells on coverslips were pulsed with 100 μ M BrdU and 18 h later BrdU incorporation was analyzed by IF. Data were expressed as % of cells. In ( b and d ), cell growth was measured 24 h later by MTT assay and data were expressed as relative increase. Quiescent mouse embryo fibroblasts (MEFs in e ) or HT1080 cells ( f ) or primary mouse fibroblasts (MFs in g and h ) on coverslips were left untreated or treated for 18 h with the indicated concentration of R1881 or DHT. Bicalutamide was added at 1000-fold excess. BrdU incorporation was analyzed as above and expressed as % of cells. In ( a–h ), means and S.E.M. are shown. n represents the number of experiments throughout the figures

Article Snippet: Karyotypically heterogeneous human fibrosarcoma HT1080 cells were a gift from P. Friedl (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: DNA Synthesis, BrdU Incorporation Assay, MTT Assay, Concentration Assay

Journal: Cell Death & Disease

Article Title: Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells

doi: 10.1038/cddis.2014.497

Figure Lengend Snippet: Rac inhibition triggers DNA synthesis and suppresses p27 Ser10 phosphorylation in NIH3T3 fibroblasts and human fibrosarcoma HT1080 cells treated with 10 nM R1881. In ( a – c ), NIH3T3 cells were used. In ( a ), quiescent cells on coverslips were left untreated or treated for 18 h with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. After in vivo pulse, BrdU incorporation was analyzed by IF and expressed as % of cells. In ( b ), quiescent cells were left untreated or treated for 30 min with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. Lysate proteins were analyzed by western blot, using antibodies against the indicated proteins. In ( c ), growing cells were transfected with non-targeting siRNA (ctrl siRNA) or Rac1 siRNA (Rac1 siRNA). Cells were co-transfected with negative control siRNA Alexa-Fluor 488 to help identification of transfected cells. After transfection, the cells were made quiescent and then left unstimulated or stimulated for 18 h with 10 nM R1881. After in vivo pulse, BrdU incorporation was analyzed by IF and expressed as % of transfected cells. Data are derived from at least 500 scored cells for each experiment. Inset in ( c ) shows the western blot with anti-Rac antibody of lysate proteins from cells transfected with Rac1 or non-targeting (ctrl) siRNA. The filter was re-probed with anti-tubulin antibody as a loading control (tub). In ( d ), quiescent HT1080 cells on coverslips were left untreated or treated for 18 h with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. BrdU incorporation was analyzed as in ( a ). Means and S.E.M. are shown

Article Snippet: Karyotypically heterogeneous human fibrosarcoma HT1080 cells were a gift from P. Friedl (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: Inhibition, DNA Synthesis, Phospho-proteomics, Control, In Vivo, BrdU Incorporation Assay, Western Blot, Transfection, Negative Control, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Selective 3-MST Inhibitor I3MT-3 Works as a Potent Caspase-1 Inhibitor

doi: 10.3390/ijms26052237

Figure Lengend Snippet: I3MT-3 exerts an inhibitory effect on inflammasome activation by targeting caspase-1 but not ASC. ( A , B ) HEK293A cells were transfected with plasmids of pro-caspase-1 and pro-IL-1β for 6 h, followed by overnight treatment with the above concentrations of I3MT-3. Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies ( A ). IL-1β release was analyzed by ELISA ( B ). Data shown are the mean ± S.D. Significant differences were determined by one-way ANOVA, followed by the Tukey–Kramer test; *** p < 0.001. ( C ) BMDMs were treated with the indicated concentrations of I3MT-3 and 100 ng/mL LPS for 4 h, and then treated with 1 mM ATP for 1.5 h. Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. ( D ) PMA-differentiated ASC KO THP-1 cells were pretreated with 50 µM I3MT-3 for 1 h and then treated with 1 μM Talabostat for 3 h. Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. ( E ) HEK293A cells were transfected with plasmids of Flag-pro-caspase-1 for 24 h and then treated with 50 μM I3MT-3 or 50 μM VX-765 for 4 h. Purified Flag-caspase-1 was incubated for 20 min at RT in assay buffer. Then Ac-WEHD-pNA Colorimetric substrate 100 µM was added, and it was incubated at 37 °C for 2 h. Activity was measured using a microplate reader and the absorbance was read at 405 nm. Data shown are the mean ± S.D. Significant differences were determined by one-way ANOVA, followed by the Tukey–Kramer test; *** p < 0.001. ( F ) HT1080 cells were pretreated with the indicated concentrations of I3MT-3 for 24 h and then treated with 50 ng/mL FasL for 4 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. ( G ) HEK293A cells were transfected with plasmids of Flag-pro-caspase-3 for 24 h and then treated with 50 μM I3MT-3 or 20 μM z-VAD-fmk for 4 h. Caspase-3 activity was measured by the Colorimetric caspase-3 assay. Data are shown as the ratio of caspase-3 activity versus the corresponding controls. Data shown are the mean ± S.D. Significant differences were determined by one-way ANOVA, followed by the Tukey–Kramer test; *** p < 0.001, N.S. not significant.

Article Snippet: Human monocytic leukemia cell line THP-1 and human fibrosarcoma cell line HT1080 were obtained from the JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank) [ ].

Techniques: Activation Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Activity Assay, Caspase-3 Assay

Journal: Scientific Reports

Article Title: The oncolytic adenovirus VCN-01 promotes anti-tumor effect in primitive neuroectodermal tumor models

doi: 10.1038/s41598-019-51014-1

Figure Lengend Snippet: Characterization of VCN-01 in PNET cell lines in vitro . ( A ) Expression of adenoviral receptors CAR, α v β 3 integrin and α v β 5 integrin in CNS-PNET cell lines PFSK-1 (left) and SK-PN-DW (right). Graph shows the percentage of stained cells for each receptor (Mean ± SD; n = 3). ( B ) PFSK-1 and SK-PN-DW (200,000 cells) infected with the GFP expressing vector AdTLRGDK at MOIs of 0, 0.1, 1, 10 or 100 PFU/cell. Graph indicates the percentage of GFP positive cells expression measured by flow cytometry at 24 h (white bars) or 48 h (black bars) after the infection (Mean ± SD; n = 3). ( C ) Detection of the viral proteins E1A and fiber in whole-cell lysates 48 h after being from PFSK-1 and SK-PN-DW from VCN-01 infected PFSK-1 and SK-PN-DW cultures. Grb2 was used as loading control protein. Blots from different parts of the same gel have been grouped to improve clarity of the image. ( D ) Viral titers in PFSK-1 and SK-PN-DW (50,000 cells/well) cultures 72 h after being infected with VCN-01 at MOIs 1 and 10. Bars represent total PFUs contained in the lysates (Mean ± SD; n = 3).

Article Snippet: PNET cell lines PFSK-1 (ATCC, Manassas, VA, USA; CRL-2060 TM ) and SK-PN-DW (ATCC, CRL-2139 TM ) were stained with unlabeled monoclonal antibodies recognizing the adenoviral receptors CAR (Merck Millipore, Temecula, CA, USA; 05–644) α v β 3 integrin (Merck Millipore; CBL544) and α v β 5 integrin (R&D Systems Inc., Minneapolis, MN, USA; MAB2528), and donkey anti-mouse IgG1-AlexaFluor488 (Thermo Fisher Scientific, Waltham, MA, USA; A-21202) as secondary antibody.

Techniques: In Vitro, Expressing, Staining, Infection, Plasmid Preparation, Flow Cytometry, Control