Fig. 2

A In vitro assays demonstrate rapid and potent effect of HTL-001 in a panel of GBM cell lines. Cells were treated with HTL-001 for 2 hours at a range of doses (15, 30, 60, 90 and 120µM). The % cell survival was assessed relative to untreated cells via an MTS assay. HTL-001 IC50 values ranged from 21-84 to 66.13µM. Error bars represent standard deviation of 8 replicates within one biological repeat. B Annexin V and 7-AAD staining demonstrates that HTL-001 induces GBM cell death via apoptosis. The % viable cells, % apoptotic cells and corresponding FACS plots C (after Annexin V and 7-AAD staining) are shown in LN18, U87 and GL-261 GBM cell lines treated with HTL-001 for 2 hours at 15, 30, 60, 90 and 120µM. Error bars represent standard deviation within one biological repeat. D Total protein lysates were extracted from untreated and treated cells and probed with either an anti-cFOS antibody, an anti-DUSP1 antibody or an anti-EGR1 antibody, via western blot analysis. All three markers of interest showed significant upregulation in protein expression after 2-hour treatments with HTL-001. E Protein lysates were extracted from untreated and treated cells and probed with an anti-caspase 3 antibody via western blot analysis. Neither caspase 3 nor cleaved caspase 3 significantly increased after 2-hour treatments with HTL-001. F Subcellular fractionation was used to extract mitochondrial and nuclear protein lysates from cells treated with HTL-001. Western blot analyses with an anti-AIF antibody was used to show a significant increase in mitochrondrial and translocated AIF (mAIF and tAIF, respectively). G Caspase induction in cells treated with HTL-001 was measured by adding Caspase-Glo 3/7 assay reagent to respected wells and measuring the luminescence of triplicate wells. There was no significant increase in caspase 3/7 activity in cells treated with HTL-001. H Calpain induction in cells treated with HTL-001 was measured by adding Calpain-Glo Protease assay reagent to respective wells and measuring the luminescence of triplicate wells. There was a significant increase in active calpain present in cells treated with HTL-001, in comparison to the control level.