Noblotting analysis. HeLa cells were stably transfected with shNHERF1 constructs (HeLa-NHERF1-KD), and CaSki cells were transiently transfected with NHERF1 siRNAs (CaSki-NHERF1-KD). b Knockdown of NHERF1 enhanced proliferation of cervical Pipamperone web cancer cells. Proliferation of HeLa-NHERF1-KD, CaSki-NHERF1-KD, and their handle cells was detected by CCK-8 at the indicated time points (repeated-measures analysis of variance, p 0.01, error bars represent imply ?s.d., n = 3). c Knockdown of NHERF1 enhanced the colony formation of cervical cancer cells. Colony formation was monitored in HeLa or CaSki cells for 7 days. Prime panel: Representative photographs on the clonogenicity. Bottom panel: Quantification on the colony formation efficiency (t test, p 0.05, error bars represent imply ?s.d., n = three). d Inhibition of NHERF1 expression enhanced cell proliferation of cervical cancer cells by CFSE assay (t test, p 0.01, error bars represent mean ?s.d., n = three). Cells had been stained with CFSE and analyzed following the protocol as described inside the “Methods”. e Overexpression of NHERF1 in cervical cancer cells was verified by immunoblotting evaluation. HeLa and CaSki cells have been transiently transfected with NHERF1 constructs, respectively, and expression of NHERF1 was verified by western blotting. f Exogenous NHERF1 expression inhibited the colony formation of cervical cancer cells. Colony formation was monitored in HeLa or CaSki cells for 7 days. Best panel: Representative photographs with the clonogenicity. Bottom panel: Quantification of the efficiency of colony formation (t test, p 0.05, error bars represent mean ?s.d., n = three). Cells proliferation was detected by CCK-8 assay in the indicated time points (repeated-measures evaluation of variance, p 0.01, error bars represent mean ?s.d., n = 3)Official journal from the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2018)9:Page 5 ofcells (Fig. 2f), and these information were constant together with the proliferation benefits from HeLa cells (Fig. S3). Taken together, these findings indicate that NHERF1 inhibits proliferation of cervical cancer cells.NHERF1 inhibits cervical cancer cell proliferation via downregulation of ACTNWe previously reported that NHERF1 downregulated ACTN4 protein expression Lenalidomide-I MedChemExpress levels by promoting ACTN4 ubiquitination and proteasomal degradation25. ACTN4 could promote cervical cancer cell proliferation26. Hence, it is actually hugely attainable that NHERF1 might inhibit proliferation of cervical cancer cells through regulation of ACTN4 protein expression. In an effort to explore this possibility, the endogenous levels of NHERF1 and ACTN4 in CaSki and HeLa cells were analyzed. We identified that CaSki expressed reasonably low levels of NHERF1 and high levels of ACTN4 compared with HeLa cells (Fig. S4A), whereas CaSki cells, as expected, exhibited higher proliferation potential than HeLa cells (Fig. S4B ), implying a possible part of NHERF1 in cervical cancer cell proliferation by means of regulation of ACTN4. To further confirm this hypothesis, proliferation of cervical cancer cells was analyzed following combined depletion of ACTN4 and NHERF1 expression. Data showed that knockdown of NHERF1 expression upregulated ACTN4 protein levels, which were constant with our previous report25, and promoted proliferation of HeLa (Fig. 3a) and CaSki cells (Fig. 3b) as compared using the control. Even so, when ACTN4 expression was knocked down by siRNA, NHERF1 had much less impact on the cervical cancer cell proliferation (Fig. 3a, b and Fig.