Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Jun 16;11(6):466. doi: 10.1038/s41419-020-2671-1

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 6 — a, b Chromatin immunoprecipitation (ChIP) assay of binding sites of KLF5 on the IGF1 promoter detected by qPCR in 22RV1 and PC-3 cells. GADPH promoter was served as a negative control. c The map of IGF1 promoter, which indicates the regions analyzed in ChIP assay. d Oligonucleotide DNA pull-down assay was performed to verify that the DNA fragment amplified by PCR could pull-down KLF5 protein in 22RV1 cells; the pull-down protein was detected by western blotting. e, f Co-immunoprecipitation (Co-IP) assay of the mutual interaction of KLF5 and HDAC1 proteins at endogenous (22RV1 and PC-3) (e) and exogenous levels (293T) (f), as detected by western blotting analysis. IgG was used as the negative control, while input was a positive control. g, h Western blotting analysis of HDAC1, KLF5, and IGF1 protein expression in 22RV1, C4-2, PC-3, and DU145 cells transfected with shKLF5 lentivirus and si-HDAC1 siRNA. i, j Expression of IGF1 and HDAC1 mRNA was negatively correlated in prostate cancer tissues, as per analysis in GSE16560 and GSE60329. k, l ChIP-qPCR assay of HDAC1 binding on IGF1 promoter in 22RV1 and PC-3 cells transfected with shKLF5 or shNC. Treatment with IgG was used as a negative control. m, n Oligonucleotide DNA pull-down assays were performed to verify that 100 pmol specific DNA fragment precipitated HDAC1 protein in NC and shKLF5 sublines of 22RV1 and PC-3 cells. No-biotin labeled DNA was used as a negative control. o, p Dual-glo luciferase assay indicated that knockdown of KLF5 in PC-3 and 22RV1 cells promoted the transcriptional activity of IGF1 promoter. q, r Dual-glo luciferase assay showed that knockdown of HDAC1 further increased the transcriptional activity of the IGF1 promoter activated by knockdown of KLF5 in 22RV1 and PC-3 cells. ns p > 0.05; *p <0.05, **p <0.01, ***p <0.001. The pGL3-IGF1#1, pGL3-IGF1#2, and pGL3-IGF1#3 indicate three different oligonucleotides located in the proximal region of the IGF1 promoter. Plasmid pGL3-Basic served as a negative control. KLF5, HDAC1, and IGF1 expression in 22RV1 and PC-3 cells treated with shKLF5 and si-HDAC1 detected by western blotting.