primary antibodies against e coli  (Bio-Rad)

Journal: Molecular cancer research : MCR

Article Title: A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients

doi: 10.1158/1541-7786.MCR-21-0255

Figure Lengend Snippet: (A, B) Ex vivo drug testing of the novel patient derived cell model (WCM197) together with the OH931, NMFH-1 and HFF (benign human fibroblasts) using abemaciclib a second line CDK4/6-inhibitor and pralatrexate an antifolate inhibitor in a 6–8-point dilution dose response log scale to determine the IC50 values, highest dose was 10uM and lowest dose was 4.7 pM with an assay time of 96 hours. Greater cytostatic and cytotoxic effect was seen in the cells with the CDKN2A/B and MTAP null status (WCM197 and OH931) compared to CDKN2A/B and MTAP wild type status (NMFH-1 and HFF). (C) The combination of pralatrexate (IC30) and abemaciclib killed all our patient cells within 48 hours compared to the vehicle and abemaciclib and pralatrexate mono-therapy. (D) WCM197 sarco-sphere formation is completely inhibited with the combination of abemaciclib and pralatrexate. (E) Drug combination of abemaciclib and pralatrexate causes massive cell swellings and cytoplasmatic vacuole formation. (F) Cleaved caspase 3 and cleaved PARP at 24, 48 and 72 hours were detected with the combination of abemaciclib and pralatrexate indicating the induction of cell death in the tumor cells after treatment.

Article Snippet: Primary antibodies against CDKN2A (1:2000, Abcam Cat# ab208349), MTAP (1:2000, Abcam Cat# ab126623), RB (1:1000, Abcam Cat# ab32513), Cleaved Caspase-3 (Asp 175) (1:1000, Cell Signaling Cat# 9664), cleaved PAPR (aSP214) (1:1000, Cell Signaling Cat# 32563) and Actin (1:2000, EMD Millipore clone 4, Cat# 1501) were incubated over night at 4°C in 5% bovine serum albumin.

Techniques: Ex Vivo, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients

doi: 10.1158/1541-7786.MCR-21-0255

Figure Lengend Snippet: (A) High throughput drug screening of all 4 cell strains (HFF, OH931, WCM197, NMFH-1) shown as a heat map (blue is sensitive to red resistant). RB1 wt and CDKN2A/B/MTAP null cells (WCM197 and OH931) show higher sensitivity to CDK4/6 inhibititors such as abemaciclib (indicated by the yellow arrows) and the antifolate pralatrexate and methotrexate (indicated by the green arrows) compared to the CDKN2A/B/MTAP wild type lines (NMFH-1 and HFF). Selective sensitivity to PLK-1 inhibitors was detected in WCM197 and OH931 cells compared to the NMFH-1 and the control line HFF, shown in orange arrows. (B) Graphs show the response of the sarcoma cells to each compound in the library as area under the curve (AUC) compared to the benign human foreskin fibroblast line (HFF) as a normal control. (C-D) Ex vivo drug validation of the two PLK-1 inhibitors (volasertib and rigosertib) confirms high sensitivity for WCM197 and OH931 cells. (E, F) WCM197 3D sarco-spheres are completely inhibited in growth under volasertib treatment and show cell death indicated by cleaved caspase 3, PARP and cleaved PARP. Cell death was monitored with cleaved caspase 3 and cleaved PARP over 96 hours with a peak at 24–72 hours.

Article Snippet: Primary antibodies against CDKN2A (1:2000, Abcam Cat# ab208349), MTAP (1:2000, Abcam Cat# ab126623), RB (1:1000, Abcam Cat# ab32513), Cleaved Caspase-3 (Asp 175) (1:1000, Cell Signaling Cat# 9664), cleaved PAPR (aSP214) (1:1000, Cell Signaling Cat# 32563) and Actin (1:2000, EMD Millipore clone 4, Cat# 1501) were incubated over night at 4°C in 5% bovine serum albumin.

Techniques: High Throughput Screening Assay, Control, Ex Vivo