(A) Representative spectrum for CAPERα identified in anti-TBX3 co-IP of HEK293 cell lysates. Mass spec analysis identified six specific CAPERα peptides, providing 8.5% sequence coverage of the …
(A) Anti-Tbx3 IB of exon 7 missense (ex7) and wt proteins expressed in HEK293 cells also transfected with anti-TBX3 shRNA. The overexpressed proteins are produced (red arrowhead). (B) anti-CAPERα …
(A–C) Representative bright field images of senescence associated β-galactosidase (SA-βG) assays of HFFs transduced with control, TBX3 shRNA A or CAPERα shRNA A. Only occasional cells in the control …
(A) RT-PCR analysis of CAPERα and HPRT transcript levels in HFFs transduced with two different retroviruses producing anti-CAPERα shRNAs (CAP sh A and B) and control shRNA virus (Ctl sh). Red …
(A) RT-PCR analysis of TBX3 and HPRT transcript levels in primary human foreskin fibroblasts (HFFs) transduced with control (Ctl sh) or TBX3 (TBX3 shA) shRNA retrovirus. (B) RT-PCR analysis of TBX3 …
. (A–B′) Representative WT and Tbx3 null MEFs cells stained for laminβ1 at passage (P) 4 (A and B) and P1 (A′ and B′); note nuclear distortion and rupture in senescing Tbx3 null MEFs as early as P1. …
(A–F) SA-βgal assays of HFFs stably transduced with control (Ctl) or p53 (Masutomi et al., 2003) or RB (Boehm et al., 2005) shRNAs subsequently transduced with CAPERα or TBX3 shRNAs. (G) % …
(A–C) RT-PCR analysis of p53 (A), RB (B) and p16 (C) transcript levels relative to HPRT after shRNA-mediated KD in HFFs. The shRNAs employed for these knockdowns were obtained from Addgene and have …
Seven regions tested upstream of CDKN2A-p16 promoter by ChIP with anti-TBX3 and anti-CAPERα antibodies. Amplicons are numbered black boxes 1–7 ‘Your Seq’ at top superimposed on window from UCSC …
ChIP-PCR of CDKN2A-p16 regulatory elements with anti-H3K27me3 in control, TBX3 or CAPERα shRNA-transduced HFFs. Locations of amplicons relative to transcription start site are noted in parentheses …
(A) ChIP-PCR of CDKN2A-p14 promoter with antibodies listed at top in control (C) and TBX3 siRNA (C′) transduced HFFs. Red arrowhead indicates loss of CAPERα binding after TBX3 knockdown. (B–E) …
(A–C) Gel showing RT-PCR analysis of TBX3, CAPERα, and HPRT expression in control, TBX3 and CAPERα siRNA-transfected HEK293 cells. The siRNAs effectively decreased transcript levels of their …
(A) Additional representative differential display gels with transcripts unchanged or independently affected by knockdown of CAPERα or TBX3 in HEK293 cells. (B) RT-PCR validating differential …
(A) UCA1 and HPRT transcripts assessed by RT-PCR in control and UCA1-overexpressing HFFs. (B and C) Representative bright field images of SA-βgal assay of cultured HFFs transfected with control and U…
pcDNA3.1 are control transfected cells and UCA1 were transfected with UCA1 expression plasmid in pcDNA3.1 (as in Figure 5A). Note increased p16 and p21 levels and hypophosphorylation of RB. TBX3, …
Input, rabbit IgG negative control ChIP, and H3K9acetylation ChIP in control “C” or UCA1 “U” transfected HFFs for gene regulatory regions as labeled at bottom (primer sequences listed in ChIP …
(A) Graphs of transcript levels assayed by RT-qPCR in HFFs transfected with control (blue) or UCA1 (red) expression plasmids and treated with Actinomycin (D). Y axis shows % mRNA level relative to …
TBX3, CAPERα = human; Tbx3, Caperα = mouse.
(A) Top panel shows Northern blot of HFF cells transfected with control plasmid pcDNA3.1 and treated with Actinomycin D for the times (hr) indicated at top. (A′) The ethidum bromide stained gel …
(A) Western blot assaying hnRNP A1 protein levels in HFFs after transfection of control or anti-hnRNP A1 siRNA. (B) Graphs of best fit lines, equations and R values for half-lives shown in Figure 6B.…
RIP-PCR of MYC and CDKN2A-p14 mRNAs shows they are bound by hnRNP A1 but these interactions are unaffected by OIS/RAS, UCA1 overexpression, or knockdown of TBX3 or CAPERα. TBX3, CAPERα = human; …
Only hnRNP A1 (A) and hnRNP D (B) bind UCA1 lncRNA, while TUG1 and H19 lncRNAs are bound by other hnRNPs. TBX3, CAPERα = human; Tbx3, Caperα = mouse.
TBX3, CAPERα = human; Tbx3, Caperα = mouse.
(A) ChIP-PCR of regions upstream of the CDKN2A-p16 transcriptional start site (position relative to TSS in parentheses) in PS and RAS HFFs; the −-3706–3308 amplicon is a negative control. OIS …
Differentially expressed genes after knockdown of CAPERα in HFFs detected by RNA-Seq.
Differentially expressed genes after knockdown of TBX3 in HFFs detected by RNA-Seq.
Determining the statistical significance of shared differentially expressed genes using the hypergeometric test, as implemented in the R statistical language (phyper).
(A) ChIP-PCR to assess chromatin marks on CDKN2A-p16 regulatory elements in PS and RAS HFFs; antibodies are listed at top. (B) ChIP-PCR of DUSP4 promoter in PS and RAS HFFs; antibodies are listed at …
Immunocytochemical assay for endogenous CAPERα (green), PML (red), and DNA (DAPI, blue) in PS and RAS HFFs. TBX3, CAPERα = human; Tbx3, Caperα = mouse.
qPCR validation of a subset of transcripts with altered expression detected by genome wide RNA-Seq on cDNA prepared from CAPERα (red) and TBX3 (blue) KD, and RAS HFFs (green). Downregulated …
ChIP-PCR with antibodies listed at top showing CAPERα/TBX3 directly binds IL6 (and HDAC9) control elements. Effects of TBX3 or CAPER KD on chromatin marks are shown compared with control KD. …