br Fig Expression of HDLBP in SCLC and normal
Fig. 3. Expression of HDLBP in SCLC and normal lung tissues. (A) Representative IHC images of HDLBP in normal lung tissues and SCLC tissues. “-” represents negative, “+” represents weak and moderate positive and “++” represents strong positive. (B) Quantification of HDLBP protein level in SCLC tumor tissues and normal lung tissues.
W. Zhou, et al. Experimental Cell Research xxx (xxxx) xxx–xxx
Fig. 4. HDLBP knock-down inhibited the malignant phenotypes of SCLC cells in vitro. (A) Immunoblotting of HDLBP in SCLC cell lines. (B) Confocal images of HDLBP in H446 cells transfected with control siRNA (Si-NC) or HDLBP siRNA (Si-1 and Si-2). The secondary antibody for HDLBP was conjugated with TRITC (Red), the cell nucleuses were stained with DAPI (Blue). (C) Immunoblotting of HDLBP in H446 cells transfected with control siRNA or HDLBP siRNA. (D,E) Proliferation curve of H446 (D) and H1688 (E) cells transfected with control siRNA or HDLBP siRNA (n=3). (F,G) Representative images (left) and quantification (right) of Transwell assays of H446 (F) and H1688 (G) cells transfected with control siRNA or HDLBP siRNA (n=3). Differences in measured variables between groups were assessed using the Student’s t-test, *P < 0.05.
migration and invasion of SCLC cells in vitro.
2.5. HDLBP promotes Haloperidol G1/S transition
Rapid proliferation is one of the most distinct characteristics of SCLC different from other cancers and aberrant change in cell cycle progression is a well-known event leading to uncontrolled cancer cell proliferation and tumor development [26–28]. Coincidently, previous reports found HDLBP was overexpressed in quick proliferation cells and our work proved HDLBP was overexpressed in SCLC tissues and could promote the proliferation of SCLC cells [23,29]. All these results sug-gested the potential roles of HDLBP in cell cycle regulation and cell proliferation in SCLC cells. So, we used flowcytometry experiment to investigate whether abnormally expressed HDLBP could affect the cell proliferation through modulating the cell cycle progression. As the result shown in http://www.sciencedirect.com/science/ article/pii/S0304383517300691 Fig. 5A and 5B, H446 cell trans-fected with HDLBP siRNA exhibited a significantly increased population of cells in G0/G1 phase but a decreased cell counts in S phase and G2/ M, which indicating a cell cycle G1/S arrest. To further confirm the results, we measured the expression of cyclin D1, a critical player promotes the G1/S transition . Consistent with the phenomenon of increased cell percentage in G0/G1 phase, the expression of cyclin D1 was also significantly inhibited as a result of HDLBP siRNA transfection (http://www.sciencedirect.com/science/article/pii/ S0304383517300691 Fig. 5C). These results confirmed that HDLBP inhibition suppressed the proliferation of SCLC cells by modulating the
cell cycle G1/S transition.
2.6. HDLBP knockdown suppresses tumor progression of SCLC cells in vivo
To further evaluate the effects of HDLBP on tumorigenicity in vivo, HDLBP stable knockdown H446 cells were constructed using Letiviours containing hairpin shRNA targeting HDBLP. We subcutaneously trans-planted these sh-HDLBP cells and other sh-NC cells (cells transfected with a control RNA) into the right and left axilla of BALB/c Nude mice respectively. Four weeks later, xenograft tumors were collected for further analysis. HDLBP knockdown notably restricted tumor forma-tion, thus resulting in smaller tumor volume and lighter tumor weight (http://www.sciencedirect.com/science/article/pii/ S0304383517300691 Fig. 6A–C). Furthermore, immunohistochemical assay suggested that Ki-67, the protein marker of cell proliferation, was much lower in sh-HDLBP xenograft tumors than that in the control group, indicating a suppression of cell proliferation in vivo (http:// www.sciencedirect.com/science/article/pii/S0304383517300691 Fig. 6D).