PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis (2021)
Figure 4

Figure 4

Influence of PRMT1 and PRMT1-targeted arginines on p14ARF protein stability.

A, B: U2OS, HeLa and HEK293 cells were transfected with Flag-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. Overexpression was analyzed by immunoblotting using α-Flag (exogenous p14ARF proteins) and α-β-TUBULIN (loading control) antibodies (A). As visualized here, p14ARF with the Flag-epitope tagged reproducibly showed a clear migration difference between wt and mutant p14ARF proteins, with the mutants (especially the RK mutant) migrating slowlier than wt protein in the SDS-PAGE. The staining signals of the p14ARF bands were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with the protein signals of p14ARF wt set to 1. Transcript levels of wt and mutant p14ARF were determined by RT-qPCR in U2OS cells (B) as well as in HeLa and HEK293 cells (Appendix Fig. S3B, S3C). Values were normalized to GAPDH expression and presented relative to wt p14ARF, mean ± SD of triplicates.

C: HeLa cells were transfected with the indicated siRNAs (five control/non-targeting siRNAs and four PRMT1-specific siRNAs). PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_2 condition set to 1. Transcript levels of p14ARF were determined by RT-qPCR (Appendix Fig. S3D).

D: HeLa cells were infected with recombinant adenovirus encoding GFP (control) or wild type PRMT1. PRMT1 overexpression and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-CDK2 (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective CDK2 signal, as specified by the numbers below the blot, with the control condition set to 1.

E-G: HeLa cells expressing a doxycycline-inducible shRNA targeting PRMT1 were treated or not with doxycycline (Dox) for 6 days. PRMT1 depletion was monitored by immunoblotting using the indicated antibodies (E). The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the -Dox condition set to 1. PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.