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. 2021 Aug 13;22(16):8718. doi: 10.3390/ijms22168718

Figure 4.

Figure 4

Effect of physalin A on Hedgehog signaling pathway. (A) Physalin A decreased the total protein level of SMO in breast mammospheres. An Anti-SMO antibody was used for immunoblotting. (B) Effect of SMO on mammosphere formation using SMO small interfering RNA (siRNA). siRNA-transfected cancer cells were incubated for 5 days with a complete Mammocults medium, and mammosphere formation efficiency (MFE) was calculated. (C) The protein levels of GLI1 and GLI2 in physalin A-treated mammospheres were assessed using specific anti-GLI1 and anti-GLI2 antibodies. (D) The effect of GLI1 protein on mammosphere formation was assessed using siRNA of GLI1. GLI1 siRNA-transfected cells were cultured for 7 days in a complete MammoCult medium and the MFE was determined. (E) Physalin A treatment reduced Hedgehog signaling-related GLI1 gene expression in the mammospheres derived from MDA-MB-231 cells. (F) After transfection of GLI1 siRNA, total RNA was extracted from breast cancer cells. The transcripts levels of c-myc, Oct4, Nanog, and Sox2 were measured by RT-qPCR using specific primers. β-actin was used as the internal control. Mean ± SD values from three independent experiments are presented. * p < 0.05, ** p < 0.01 vs. control.