Zbtb7a

Two human urinary bladder cancer cell lines (T24 and UM-UC-3) that isolated from high-grade and late-stage tumors were used as MIBC models in this study [16 (link)]. T24 and UM-UC-3 were cultured in Dulbecco's Modified Eagle Medium (GIBCO-Invitrogen) containing 10% fetal bovine serum (ExCell Bio).The following antibodies were used in our study: Actin (Santa Cruz Biotechnology, sc-1616,1:1000), ZBTB7A (Santa Cruz Biotechnology, SC-33683, 1:1000), and HIC1 (Proteintech, 24949-1-AP, 1:1000), Mouse anti-Armenian hamster IgG-HRP (Santa Cruz Biotechnology, sc-2789, 1:2000) for ZBTB7A, HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2, 1:10000) for HIC1, HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1, 1:10000) for Actin. The primary antibodies were incubated for two hours at room temperature, while the secondary antibodies were incubated for one hour.

The BC tissue microarrays purchased from Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), contained 30 pairs of cancer tumors together with matched adjacent normal tissue. IHC staining was performed for ZBTB7A (Santa Cruz Biotechnology, SC-33683, 1:200) and HIC1 (Proteintech, 24949-1-AP, 1:200) on the same paraffin-embedded tissue blocks used for clinical diagnosis. IHC was performed using the avidin–biotin complex method (Vector Laboratories), including heat-induced antigen-retrieval procedures.
The degrees of immunostaining were reviewed and scored by two independent pathologists. The proportion of the stained cells and the extent of staining were employed as evaluation criteria, with at least 1000 tumor cells evaluated in each case. While one score was assigned based on the percentage of positive cells, another was assigned based on the staining intensity, and the final score was calculated by multiplying the two previous scores.