Journal: Cancer Medicine

Article Title: A High‐Throughput Drug Repurposing Strategy to Treat TBX2 and/or TBX3 Dependent Cancers

doi: 10.1002/cam4.70303

Figure Lengend Snippet: Niclosamide, piroctone olamine and pyrvinium pamoate regulation of TBX2 and TBX3 and the effect of tacrolimus on another Tet‐On inducible system. (A) Endogenous TBX2 and TBX3 levels in 501mel parental cell lines following 24 h treatment with 10 μM drug (2.5 μM for pyrvinium pamoate due to cell sensitivity) or vehicle determined by western blotting using antibodies to TBX2 and TBX3. (B) Quantitative RT‐PCR performed on reverse transcribed RNA extracted from Tet‐On inducible Cas9 stem cells induced with 20 ng/mL doxycycline (dox) for 24 h followed by treatment with 10 μM tacrolimus, piroctone olamine and vehicle for 24 h. Primers specific to Cas9 were used, mRNA levels were normalised to β‐actin and expressed relative to the uninduced control. (C) Western blotting showing levels of TBX2 and TBX3 in ERMS (RD cell line) and ARMS (RH30 cell line) cells after 24 h of 10 μM drug (2.5 μM pyrvinium pamoate) or vehicle treatment. (D) Quantitative RT‐PCR performed on reverse transcribed RNA extracted from 501mel, RD and RH30 cells treated for 24 h treatment with 10 μM drug (2.5 μM for pyrvinium pamoate due to cell sensitivity) or vehicle. Primers specific to TBX2 and TBX3 were used, mRNA levels were normalised to GUSB and expressed relative to the vehicle control. Data was analysed using GraphPad Prism 6.0 and a parametric unpaired t‐test was performed where, *p < 0.05, **p < 0.01, ***p < 0.001 an ****p < 0.0001. (E) Table showing the EC 50 values from MTT cell viability assays of 501mel, RD and RH30 cells treated with a range of selected ‘Hit’ drug concentrations and vehicle as indicated for 72 h.

Article Snippet: RD human embryonal rhabdomyosarcoma (ERMS) cells (ATCC CCL‐136) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich).

Techniques: Western Blot, Quantitative RT-PCR, Reverse Transcription, Control

Journal: Cancer Medicine

Article Title: A High‐Throughput Drug Repurposing Strategy to Treat TBX2 and/or TBX3 Dependent Cancers

doi: 10.1002/cam4.70303

Figure Lengend Snippet: The effect of selected ‘hit’ drugs on TBX2 and TBX3 levels in melanoma, ERMS and ARMS cell lines. (A) Representative light microscopy images (200X; EVOS XL AMEX1000 Core Imaging System) of 501mel, RH30 and RD after 24 h of 10 μM drug (2.5 μM pyrvinium pamoate) or vehicle treatment. (B) Senescence‐Associated (SA)‐β‐Galactosidase staining of 501mel, RD and RH30 cells treated with either vehicle, or EC 50 niclosamide, piroctone olamine and pyrvinium pamoate for 72 h after which cells were incubated with the SA‐β‐Gal chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl β‐D‐galactopyranoside (X‐Gal) at pH 6 and SA‐β‐Gal positive cells were identified by a distinct blue colour. Representative brightfield microscopy images (200X; EVOS M5000 Imaging System; scale bars = 100 μm) are shown. (C) Western blot analyses of protein harvested from 501mel, RD and RH30 cells treated vehicle or EC 50 concentrations of niclosamide, piroctone olamine and pyrvinium pamoate for 72 h and incubated with the PARP specific antibody. (D) 501mel, RD and RH30 cells were pre‐treated with 20 nM siControl, siTBX2 or siTBX3 24 h prior to the addition of EC 50 concentrations of niclosamide, piroctone olamine and pyrvinium pamoate for 48 h after which the MTT cell viability assay was performed. p38 was used as a loading control and densitometry readings were obtained using ImageJ and protein expression levels are represented as a ratio of protein of interest/β‐actin normalised to the vehicle control sample (where possible). Blots are representative of at least two independent repeats. Graphs show mean cell viability as a percentage of vehicle control ± SEM for each concentration of drug determined from three independent experiments performed in quadruplicate. Data was analysed using GraphPad Prism 6.0 and a parametric unpaired t‐test was performed where, * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Article Snippet: RD human embryonal rhabdomyosarcoma (ERMS) cells (ATCC CCL‐136) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich).

Techniques: Light Microscopy, Imaging, Staining, Incubation, Microscopy, Western Blot, Viability Assay, Control, Expressing, Concentration Assay