Figure 8:

(A) Autophagy related protein and mRNA expression levels of selected genes in cervical cancer cells treated with Nef: HeLa and SiHa cells were treated with Nef and allowed to grow for 48 hrs, following which cells lysed in protein lysis buffer. Proteins were separated on 10–12% SDS-PAGE and transferred to nitrocellulose membrane, probed with beclin-1, atg-4, 5&12, LC-3 and P62 antibodies. Increased expression of autophagy related proteins, which indicated that Nef induced autophagy in cervical cancer cells. Images shown are the protein bands detected by chemiluminescence. (B) Quantitative data of relative protein expression levels as determined by ImageJ software. P<0.01 compared with the control group. (C) The mRNA isolated from HeLa and SiHa cells treated with Nef (25μM) was converted to cDNA using High-capacity complementary DNA (cDNA) reverse transcription kit. Quantification of the expression levels of target genes was conducted using SYBR Green method with both forward and reverse primers. All the transcript levels were normalized using β-actin expression levels. Real time PCR data revealed that HPV early gene (E6 and E7) levels were decreased by Nef treatment compared to untreated controls. On the other hand, Nef increased the expression levels of autophagy specific genes (LC-3, beclin-1) compared to untreated controls. β-actin served as the reference house keeping control. Data are presented as mean ± standard deviation (SD) of three independent experiments. *p < 0.01 or #p<0.01 compared with the control group.