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. Author manuscript; available in PMC: 2018 Sep 15.
Published in final edited form as: Cancer Res. 2017 Jul 7;77(18):5011–5025. doi: 10.1158/0008-5472.CAN-16-2704

Figure 5. PARG overexpression rescues HuR’s regulation of PARPi response.

Figure 5

(A) Cell survival of MIA PaCa-2 cells co-transfected with HuR siRNA and PARG overexpression plasmid and treated with olaparib for 7days. (B) PARG rescue and HuR, PARP-1 and PAR expression validated, with GAPDH (total protein extract) and Histone H3 (nuclear chromatin- tethered fraction) as the loading controls. (C) ELISA to quantitate relative PARylation with PARG rescue in HuR silenced MIA PaCa-2 cells. (D) Immunofluorescence of HuR (green) in MIA PaCa-2 cells treated with veliparib for 12hr, with or without a 6hr pre-treatment of small-molecule HuR inhibitor, MS-444. Nuclei stained with DAPI (blue). Magnification 40X. (E) Relative PARylation and immunoblotting of total protein lysates of MIA PaCa-2 cells treated with increasing dosage of MS-444, in the presence of veliparib for 12h. (F) Cell survival of MIA PaCa-2 and Capan-1 cells treated with indicated doses of veliparib, with or without 5μM/L MS-444. (G) Luciferase activity in MIA PaCa-2 cells transfected with luciferase reporter constructs with PARG 3′UTR and incubated in the presence of MS-444 for 24 hr.