Panobinostat

SAOS2, SAOS2 LM7 and K7M2 cells were seeded 5 × 10³ cells per well in 96-well plates and in triplicate were treated with a dose range of panobinostat (Selleckchem, Houston, Texas, Cat #S1030; 1.8, 2.4, 3.3, 4.5, 6.0, 8.1, 11.0, 14.8 and 20 nM) or a dose range of romidepsin (Selleckchem Cat #S3020; 5.9, 8.8 13.2, 19.8, 29.6, 44.4, 66.7, 100 and 150 nM). For dual treatment with panobinostat and Carfilzomib (Selleck Cat #S2853) K7M2 and SAOS2 cells were plated at 5 × 10³ cells in 96-well plates and treated with panobinostat at a dose range (1.4, 1.8, 2.5, 3.3, 4.5, 6.1, 8.2, 11.1 and 15 nM) or Carfilzomib at a dose range (1.9, 2.6, 3.5, 4.7, 6.3, 8.5, 11.5, 15.6 and 21 nM) or combination of both drugs. IC₅₀ values were determined at 48 hours posttreatment using cell titer blue assay (Promega, Madison, Wisconsin, Cat # G8080). To determine the tubastatin (Selleckchem Cat # S2627), IC₅₀ value K7M2 cells were seeded at 2 × 10⁴ in 96-well plates and treated with the following concentrations (0, 1, 5, 10, 15, 100, 250, 500, 1250, 2500, 5000 and 10 000 nM). All treatments were done in triplicate and cell growth was assayed at 48 hours using MTS CellTiter 96 cell proliferation assay (Promega Cat # G5421). The impact of panobinostat and romidepsin on PDX derived cell lines was examined in a publicly available dataset (https://braid.stjude.org/masttour/).^{15 (link)}

RNAseq was performed as above in SU-DIPG-IV and SU-DIPG-VI cell lines following exposure to 1 µM panobinostat (Selleck Chemicals) for 48 hours. RNAseq was also performed in SU-DIPG-IV, SU-DIPG-VI and JHH-DIPG1 cell lines following exposure to 0.1 µM panobinostat (Selleck Chemicals) for 24 hours in triplicate. Cells were then lysed and RNA extracted in TRIzol solution.
Post-panobinostat RNAseq data analysis: There was a good concordance for the ratio of panobinostat-treated to vehicle-treated cell gene expression and the distribution of RPKM values were similar in both cell lines and with the two conditions. In general, the variability in terms of ratio was low, usually below 1.