caspase 3 7 reporter system  (Sartorius AG)

Journal: Slas Discovery

Article Title: Application of a High-Content Screening Assay Utilizing Primary Human Lung Fibroblasts to Identify Antifibrotic Drugs for Rapid Repurposing in COVID-19 Patients

doi: 10.1177/24725552211019405

Figure Lengend Snippet: Secondary hit triage assays. ( A ) Concentration response of the six hit compounds on αSMA expression measured as a percentage of TGFβ (DMSO) positive control (see for representative high-content image). ( B ) Concentration response of the six hit compounds on fibroblast proliferation as measured by the percentage of the TGFβ (DMSO) positive control. ( C ) Concentration response of the six hit compounds on fibroblast apoptosis at 48 h post-compound addition as measured by the activation of caspase 3/7 (see for representative image). ( D ) Concentration response of the six hit compounds on scratch wound healing as measured by wound confluence at 24 h presented as a percentage of DMSO control (see for representative image).

Article Snippet: The following day, the compounds or DMSO (0.1% v/v final concentration) control were added, followed by an apoptosis reporter (IncuCyte Caspase 3/7 Green; Sartorius, 4440; 5 µM), as previously described.

Techniques: Concentration Assay, Expressing, Positive Control, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: CRISPR-Cas9 CD133-knockout sensitizes BAKR-KO cells to trametinib-induced apoptosis via reduced BCL-2 levels. CD133 expression was compared between BAKR, CD133-knockout BAKR-KO, and sgRNA control BAKR-SC cells. ( A ) RT-PCR and ( B ) immunoblot analysis confirm depletion of CD133 RNA and protein, respectively, in BAKR-KO, compared to BAKR-SC and BAKR cells. ( C ) BAKR-SC and BAKR-KO cells were exposed to increasing trametinib concentrations for 72 h and subjected to immunoblot analysis with antibodies to the active cleaved form of caspase-3 (p17). Immunoblots were stripped of antibodies and re-probed with antibodies to cleaved PARP, BCL-2, and β-Actin for loading control. Densitometric analysis comparing intensities of protein bands relative to bands with the highest intensity is shown in immunoblots.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: CRISPR, Knock-Out, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: CRISPR-Cas9 knockout of CD133 expression in parental BAKP melanoma cells ( A ) increases caspase-3 mediated apoptosis ( B , C ) after exposure to trametinib. ( A ) Immunoblot analysis with anti-CD133 in BAKP-SC vs. BAKP-KO cells (left panel) and POT SC vs. POT-KO cells (right panel). ( B ) BAKP-SC and BAKP-KO cells were treated with indicated concentrations of trametinib (upper panels) or dabrafenib (lower panels) and subjected to Annexin FITC/PI apoptosis assays. Trametinib-treated CD133-depleted BAKP-KO cells display enhanced apoptosis relative to control BAKP-SC cells, an effect that was not seen with dabrafenib. ( C ) Fluorometric caspase-3 activity assays were performed 72 h after exposure of BAKP-SC and BAKP-KO cells to 100 nM trametinib. Results shown are the means ± SD of three biological replicates of a representative experiment; essentially the same results were obtained in three independent experiments. ** represents p < 0.01.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: CRISPR, Knock-Out, Expressing, Western Blot, Control, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: CRISPR-Cas9-mediated knockout of CD133 expression in BAKP ( A ) and POT ( B ) cells increases BAX activation, and PARP cleavage following trametinib treatment, sensitized by reduced basal levels of pro-survival BCL-xL, pAKT, and pBAD. ( A ) BAKP-KO, BAKP-SC cells, as well as ( B ) POT-SC and POT-KO cells were treated with 100 nM trametinib for 24 and 48 h, followed by immunoblot analysis with antibodies to cleaved PARP, cleaved active form of caspase-3, active BAX, BCL-xL, and β-actin as loading control. CD133 knockout in trametinib-exposed both BAKP-KO ( A ) and POT-KO ( B ) cells increases pro-apoptotic active BAX and decreases anti-apoptotic BCL-xL levels ( A , B ), sensitizing cells to apoptosis. Immunoblot analysis of pAKT, AKT, p-BAD, Bad, and β-actin as loading control in BAKP-KO ( C ) and POT-KO cells ( D ), compared to the SC controls. Densitometric analysis comparing intensities of protein bands relative to bands with the highest intensity is shown in immunoblots.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: CRISPR, Knock-Out, Expressing, Activation Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: Dox-induced CD133 expression in BAKP ( A , B ) and POT ( C ) cells, as verified by immunoblot analysis, suppress caspase-3 mediated apoptotic cell death ( D , F ) and increases cell viability ( E ) after trametinib treatment (dose-response experiments). ( A ) Cells were incubated with Dox for the indicated times and subjected to immunoblot analysis with antibodies to CD133 and β-actin for normalization. ( B ) Immunoblot analyses with anti-CD133 of BAKP-CD133 and vector control BAKP-rtTA3 cells treated with increasing trametinib doses +/− Dox, as well as ( C ) POT-CD133 cells incubated +/− Dox for 72 h. ( D ) BAKP-CD133, BAKP-rtTA3 empty vector control, and POT-CD133 cells were exposed to increasing doses of trametinib for 72 h, and subjected to Annexin-FITC/PI apoptosis assays; apoptosis (%; D ) and cell viability (%; E ) of cells are shown. ( F ) Fluorescent FLICA Caspase 3/7 activity assays reveal a significant decline in caspase 3 activity in trametinib-treated CD133-expressing BAKP cells (+Dox), compared to uninduced cells (−Dox); representative merged images of red fluorescent FLICA- and GFP-positive cells (10×) (left panel); quantification of FLICA-positive cells (with active caspase-3, right panel). ( G ) Immunoblot analyses with antibodies to CD133, BCL-xL, and βActin of BAKP-CD133 treated with increasing trametinib doses +/− Dox. BAKP-CD133 cells were constitutively expressing GFP. Results shown are the means ± SD of three biological replicates of a representative experiment of three independent experiments. *, **, ***, **** represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Densitometric analysis comparing intensities of protein bands relative to bands with the highest intensity is shown in immunoblots.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: Expressing, Western Blot, Incubation, Plasmid Preparation, Control, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: Dox-inducible CD133 expression attenuates trametinib-induced apoptosis in BAKP-CD133 ( A , B ) and POT-CD133 cells ( C ), as evidenced by reduced PARP cleavage, proteolytic activation of caspases-3 and -9, and activation of BAX ( A ), and upregulation of anti-apoptotic proteins BCL-xL, BCL-2, p-BAD, and p-AKT ( B ), in response to trametinib treatment. ( A ) BAKP-CD133 cells were exposed to 100 nM trametinib for 24 or 48 h +/− Dox, followed by immunoblot analysis with antibodies to apoptotic markers cleaved PARP, cleaved caspase-3, cleaved caspase-9, and active Bax. BAKP-CD133 ( B ) and POT-CD133 ( C ) treated with 100 nM trametinib +/− Dox were subjected to immunoblot analysis with anti-apoptotic proteins BCL-xL, BCL-2, p-BAD, p-AKT, AKT, and β-actin for normalization. Densitometric analysis comparing intensities of protein bands relative to bands with the highest intensity is shown below each band in immunoblots.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: Expressing, Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells

doi: 10.3390/ijms23042333

Figure Lengend Snippet: PI3K/Akt/ Bcl2 family pro-survival signaling pathway in CD133-positive melanoma initiating stem cells (MICs). CD133 may activate a survival pathway where (1) increased phosphorylation of AKT induces (2) phosphorylation and inactivation of BAD, (3) decreases the active form of BAX, and (4) reduces caspase activation and caspase-mediated PARP cleavage, indicating apoptosis suppression leading to drug resistance in melanomas.

Article Snippet: Cells (50,000 cells per well) were plated in 6-well plates, and triplicate wells were incubated for 24 h with or without Dox in the presence or absence of trametinib (100 nM), together with a FLICA caspase 3/7 detection reagent with a sulforhodamine group reporter (Image-iT Live Red Caspase 3/7 Detection kit; ThermoFisher, Waltham, MA, USA), in an EVOS FL Auto Imaging System (ThermoFisher, Waltham, MA, USA) with an onstage incubator.

Techniques: Phospho-proteomics, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Taurine Attenuates the Hypotaurine-Induced Progression of CRC via ERK/RSK Signaling

doi: 10.3389/fcell.2021.631163

Figure Lengend Snippet: Taurine reverses the promoting effects of hypotaurine on CRC progression. (A) The viability of CRC cell lines cultured for five days in serial concentrations of taurine, and each IC50 value were shown by Alamar Blue assay (means ± SEM). (B) The proliferative rate of CRC cell lines after treatment with taurine (10 mM) and/or hypotaurine (10 mM) (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (C) Quantification of clones formed by the indicated CRC lines after treatment with taurine (5 mM) and/or hypotaurine (5 mM) for 3 weeks (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (D) The proapoptotic effects of taurine (10 mM) and/or hypotaurine (10 mM) on CRC cells were detected by caspase 3/7 reporter assay (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (E,F) The cellular migratory effects of taurine (10 mM) and/or hypotaurine (10 mM) in CRC cells were evaluated by wound-healing assay and quantified. (means ± SEM) (one-way ANOVA followed by Tukey’s post- hoc test, * P < 0.05, *** P < 0.001). (G,H) The cellular invasive effects of taurine (10 mM) and/or hypotaurine (10 mM) in CRC cells were assessed using the transwell invasion assay and quantified (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: We purchased a caspase 3/7 reporter kit (G8091) from Promega (Madison, WI, United States).

Techniques: Cell Culture, Alamar Blue Assay, Clone Assay, Reporter Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Taurine Attenuates the Hypotaurine-Induced Progression of CRC via ERK/RSK Signaling

doi: 10.3389/fcell.2021.631163

Figure Lengend Snippet: An ERK/RSK inhibitor reverses the promoting effects of hypotaurine on tumor progression and EMT in CRC cells. (A) The effects of ERK/RSK inhibitor (SCH772984) on the phosphorylation (denoted p-) status of ERK and RSK were detected with western blotting analysis. (B) The proliferative rates of CRC cell lines after treatment with SCH772984 (2 μM) and/or hypotaurine (10 mM) (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Quantification of clones formed by the indicated CRC lines after treatment with SCH772984 (1 μM) and/or hypotaurine (5 mM) for 3 weeks (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (D) The proapoptotic effects of SCH772984 (2 μM) and/or hypotaurine (10 mM) on CRC cells were observed with caspase 3/7 reporter assay (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (E) The cellular migratory effects of SCH772984 (2 μM) and/or hypotaurine (10 mM) on CRC cells were determined by wound-healing assay and quantified. (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (F) The cellular invasive effects of SCH772984 (2 μM) and/or hypotaurine (10 mM) on CRC cells were ascertained by transwell invasion assay and quantified (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (G,H) The effects of SCH772984 and/or hypotaurine on EMT markers in CRC cells were discerned using qRT-PCR (means ± SEM) (one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: We purchased a caspase 3/7 reporter kit (G8091) from Promega (Madison, WI, United States).

Techniques: Western Blot, Clone Assay, Reporter Assay, Wound Healing Assay, Transwell Invasion Assay, Quantitative RT-PCR