Figure 2. Evaluation of colony suppression of Gefitinib alone or combined with metformin on 3 bladder cancer cell lines.

(A–C) Clonogenic assay was assessed after 7 day Gefitinib treatment alone or combined with metformin and wells were stained with crystal violet at the end of the experiment. (A) Clonogenic assay in MB49 was conducted with the treatment of 2 mM metformin, 0.4 μM Gefitinib and their combination. Above: The full view of wells were taken through stereomicroscope and images were taken through an inverted microscope with ×10 magnification. Below: the quantification of colony was determined by microplate area scan at OD 550 nm, Results are presented as the median of 5 independent experiments (*P < 0.05, ^#P < 0.01 vs control). (B,C) Clonogenic assay was conducted with the treatment of 2 mM metformin, 8 μM Gefitinib and their combination in T24 and UMUC3, respectively. The full view of wells and their quantification were obtained through the same method as described in MB49. Results are presented as the median of 5 independent experiments (*P < 0.05, ^#P < 0.01 vs control). (D–F) Colony formation assay was carried out with two hour treatment at labeled concentrations, twice per week for two weeks and stained with crystal violet at the end of the experiment. (D) Clonogenic assay in MB49 was conducted with the treatment of 25 mM metformin, 5 μM Gefitinib and their combination. The full view of wells and their quantification were obtained through the same method as described in MB49. Results are presented as the median of 5 independent experiments (*P < 0.05, ^#P < 0.01 vs control). (E,F). Clonogenic assay was conducted with the treatment of 25 mM metformin, 100 μM Gefitinib and their combination in T24 and UMUC3, respectively. The full view of wells and their quantification were obtained through the same method as described in MB49. Results are presented as the median of 5 independent experiments (*P < 0.05, ^#P < 0.01 vs control).