Fig 2: Localization of CSF-1R and TSC-22HeLa cells were non-transfected (A) or co-transfected with pcDNA4-TSC-22 and CSF-1R (B). ICC was performed using CSF-1R (green) and TSC-22 (red) antibodies, and their co-localization was represented by a yellow color. Cell images were taken using a confocal fluorescence microscope (magnification ×400).

Fig 3: Pro-apoptotic role of TSC-22 in cervical cancer cells(A) HeLa and Caski cells were plated in 24-well plates and transiently transfected with pcDNA4 or pcDNA4-TSC-22. The number of surviving cells was counted under a microscope. (B) HeLa cells were transiently transfected with pcDNA4 or pcDNA4-TSC-22. After the indicated time points, AnnexinV/PI double staining was performed to quantify apoptosis of HeLa cells using flow cytometry. (C) Protein level of cleaved PARP was analyzed using western blotting of control and TSC-22 overexpressing cells. (D) TSC-22- or CSF-1R-transfected HeLa cells were re-plated in 100-mm dishes and cultured for 14 days. The number of crystal violet stained colonies was counted.

Fig 4: Inhibition of AKT and ERK signaling pathways and transcriptional activity of NF-?B by TSC-22(A) HeLa cells were transiently transfected with pcDNA4-TSC-22, then harvested at the indicated time points and analyzed by western blot. The expression levels of pERK, ERK, Elk-1, Egr-1, pAKT, AKT, pGSK-3ß, and GAPDH were measured. (B) HeLa cells were transiently transfected with TSC-22 in a dose-dependent manner. The activity of the NF-?B promoter was measured using a luciferase reporter gene assay. The firefly luciferase and Renilla luciferase values were determined. Firefly luciferase levels were normalized to Renilla luciferase levels. (C) TSC-22-overexpressing cells were harvested at 24-hour intervals and NF-?B protein levels were evaluated using western blotting.

Fig 5: Mapping of the binding regions between TSC-22 and CSF-1RLeft schematic diagram displays the cDNA deletion constructs of CSF-1R and TSC-22. Right panel shows the results of protein-protein interaction using growth and ß-galactosidase assays. Positive transformants are indicated by the formation of colonies on the plate.