Journal: Cancer Biology & Therapy

Article Title: The Cdk inhibitor dinaciclib as a promising anti-tumorigenic agent in biliary tract cancer

doi: 10.1080/15384047.2024.2439057

Figure Lengend Snippet: Dinaciclib reduces the expression of the anti-apoptotic protein Mcl-1 and the phosphorylation at Ser64. Effect of dinaciclib on (A) Mcl-1 and (B) phospho-Mcl-1 (Ser64) protein expression in KKU-100, OCUG-1 and OZ cells. Biliary tract cancer (BTC) cells were either treated with the corresponding IC 50 (KKU-100: 8 nM, OCUG-1: 33 nM, OZ: 7 nM), 50 nM (KKU-100) or 100 nM (OCUG-1 and OZ) dinaciclib for 24, 48 and 72 h, respectively. Protein expression was quantified via normalization to corresponding loading controls (figure S7), which were further referred to untreated control cells [x-fold to untreated control = UTC]. Data are presented as mean values ± SEM, n = at least 3 and significances were tested for each dinaciclib concentration against controls using a one-way analysis of variance (ANOVA), Dunnett **** p < .05, * p < .01, ** p < .001 and *** p < .0001.

Article Snippet: Supernatants were mixed 1:1 with 2X novex TM tris-glycine sodium dodecyl sulfate sample buffer (Thermo Fisher Scientific; Vienna, Austria) and incubated at 95°C for 5 min. About 7.5 × 10 4 cells per slot were loaded onto stain-free gradient gels (4–20% Mini-PROTEAN® TGXTM Gel (Bio-Rad, Hercules, US)) and run at 100 V for 60–80 min. Nitrocellulose membrane blots were incubated with primary antibodies: anti-Cdk1 (#77055, monoclonal, rabbit, 1:1000), anti-Cdk2 (#18048, monoclonal, rabbit, 1:1000), anti-Cdk5 (#2506, monoclonal, rabbit, 1:1000), anti-Cdk9 (#2316, monoclonal, rabbit, 1:1000), anti-AKT (#9272, polyclonal, rabbit, 1:1000), anti-EGFR (#4267, monoclonal, rabbit, 1:1000), anti-PTK2 (#71433, monoclonal, rabbit, 1:1000), anti-STAT3 (#9139, monoclonal, mouse, 1:1000), anti-ERK (#9102, polyclonal, rabbit, 1:1000), anti-Mcl-1 (#5453T, monoclonal, rabbit, 1:1000), anti-phospho-Mcl-1 (Ser64) (13297S, monoclonal, rabbit, 1:1000), anti-phospho-EGFR (Tyr1068) (#2234, monoclonal, rabbit, 1:1000), anti-phospho-STAT3 (Tyr705) (#9145S, monoclonal, rabbit, 1:1000), anti-phospho-ERK (Thr202/Tyr204) (#9106S, monoclonal, mouse, 1:1000); all purchased from Cell Signaling Technology, Frankfurt, Germany) overnight at 4°C.

Techniques: Expressing, Phospho-proteomics, Control, Concentration Assay

Journal: bioRxiv

Article Title: Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics

doi: 10.1101/2020.04.10.036319

Figure Lengend Snippet: (A) Wild-type (WT) or BCR-ABL + B-ALL cells deficient (KO) for ERN1 (encodes IRE1), ATF6 , or Eif2ak3 ( encodes PERK ) were treated with 1.25µM DHA for 9h and MCL-1 expression was determined by immunoblotting with indicated antibodies. (B) Heme levels were measured in BCR-ABL + B-ALL cells treated with DMSO, 62.5µM (Aminolevulinic acid) ALA, or 62.5µM (succinylacetone) SA for 24h.

Article Snippet: Antibodies used were: anti-MCL-1 (Rockland Immunochemical), anti-human MCL-1, anti-PERK, anti-ATF6, anti-IRE1, anti-CHOP, anti-ATF4, anti-BCL-XL, anti-Phospho-eIF2α, anti-eIF2α (Cell Signaling), anti-HRI, and anti-Actin (Millipore) Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunochemical.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics

doi: 10.1101/2020.04.10.036319

Figure Lengend Snippet: ( A ) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (B) Wild-type (WT) or Eif2ak1 -KO (lacking HRI) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (C) Purified HRI protein was incubated with 20 µM heme, 20 µM DHA, or a combination of the two at room temperature for 30 min. Absorbance was then measured at 420 nm to detect the presence of a Soret peak which is indicative of protein binding. Data are the average of three experiments and error bars are SEM. Unpaired t-test indicates significance between HRI+Heme vs. HRI+Heme+DHA p<0.01**. (D) Wild-type or Eif2ak1 -KO cells were treated with the indicated drugs for 9h and then pulse-labeled with 35 S-methionine-cysteine for 1h. Autoradiography was used to determine new protein synthesis. Cycloheximide (CHX) served as a positive control for translation inhibition. Total protein was determined by Ponceau staining. MCL-1 expression was determined by immunoblotting. (E) WT or Eif2ak1 -KO BCR-ABL + B-ALL cells were treated with ABT-263 or ABT-199 (0, 40, 80, 160 nM) alone or in combination with the indicated concentrations of DHA for 24h. Viable cells were measured using Annexin-V and propidium iodide staining. Data are the average of three experiments and error bars are SEM. Two-way ANOVA with Bonferroni multiple comparison indicates significance P<0.0001**** between the DHA alone (0 nM ABT-263 or ABT-199) and 160 nM ABT-263 or ABT-199 at indicated doses of DHA. The combination of DHA+ABT-263 showed a statistically less synergistic response in Eif2ak1 -KO (α=3.15, p=5.67e-06) as compared to wild-type (α=5.41, p=7.5e-08) BCR-ABL + B-ALL cells (p<10 -5 ). The combination of DHA+ABT-199 showed a significantly less synergistic response in Eif2ak1 -KO (α=5.4, p=1.16e-83) as compared to wild-type (α=7.96, p=2.7e-32) BCR-ABL B-ALL cells (p<0.057).

Article Snippet: Antibodies used were: anti-MCL-1 (Rockland Immunochemical), anti-human MCL-1, anti-PERK, anti-ATF6, anti-IRE1, anti-CHOP, anti-ATF4, anti-BCL-XL, anti-Phospho-eIF2α, anti-eIF2α (Cell Signaling), anti-HRI, and anti-Actin (Millipore) Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunochemical.

Techniques: Expressing, Western Blot, Purification, Incubation, Protein Binding, Labeling, Autoradiography, Positive Control, Inhibition, Staining

Journal: bioRxiv

Article Title: Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics

doi: 10.1101/2020.04.10.036319

Figure Lengend Snippet: (A) Densitometry of MCL-1 expression in bone marrow cells harvested from BTdCPU treated mice (related to ). Data are the average of 3 separate animals and error bars are SEM. Unpaired t-test indicates significance between vehicle and BTdCPU p<0.01**. (B) Response surface modeling was used to assess synergy in cells isolated from vehicle or combination treated mice when treated with BTdCPU and ABT-263 ex vivo .

Article Snippet: Antibodies used were: anti-MCL-1 (Rockland Immunochemical), anti-human MCL-1, anti-PERK, anti-ATF6, anti-IRE1, anti-CHOP, anti-ATF4, anti-BCL-XL, anti-Phospho-eIF2α, anti-eIF2α (Cell Signaling), anti-HRI, and anti-Actin (Millipore) Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunochemical.

Techniques: Expressing, Isolation, Ex Vivo