Fig 2: MC1R-cAMP signaling enhances chromatin-bound protein levels of ATR, XPA and AKAP12 following cisplatin treatment. (A) Wild-type primary human melanocytes (PHM). (B) MC1R WT-expressing HEK293 cells or (C) MC1R R151C-expressing HEK293 cells were pre-treated with either forskolin (10 µM), MSH (100 nM), MSH (100 nM) + ASIP (100 nM) or MSH (100 nM) + HBD3 (100 nM) 30 minutes before cisplatin (100 µM) or mock treatment for 1 hr. Chromatin extracts were isolated and probed for either anti-XPA, anti-ATR, anti-AKAP12 or anti-H2A by Western blotting. ATR and XPA bands were cropped from the same blot. AKAP12 bands were cropped from a separate blot.

Fig 3: AKAP12 enhances XPA localization to UV-induced DNA damage. (A) CRISPR-deleted AKAP12 cells expressing HA-tagged AKAP12-WT or HA-tagged AKAP12?PKA were pre-treated with vehicle or forskolin (10 µM) for 30 min and mock treated or UVB irradiated (10 J/m2). Immunoblots were performed on chromatin-fractions with anti-XPA. Inputs represent total chromatin H2A levels and 10% of whole cell lysate levels of XPA. (B) XPA-[6-4]-PP interactions were determined by proximity ligation using anti-XPA and anti-[6-4]-PP antibodies. CRISPR-deleted AKAP12 HEK293 cells were transfected with either wild-type AKAP12 or AKAP12?PKA and treated with either vehicle or forskolin (10 µM) and exposed to UVC (10 J/m2). Green detection events signify juxtaposition between AKAP12 and XPA in maximum intensity projection images 30 min after UV exposure. Nuclei were stained with DAPI (blue). Bar represents 50 µm. (C) Quantification of the XPA-[6-4]-PP colocalization shown in panel C. At least 100 cells were counted from representative fields from two separate experiments and extent of interaction is expressed as fold-change compared to the damage-null cells obtained from maximum intensity images from focal plane z-stacks. Values not sharing a common letter were significantly different as determined by one-way ANOVA; P = 0.05. Data are expressed as mean ± SEM. (D–F) Chromatin-associated XPA levels in CRISPR-deleted AKAP12 HEK293 cells expressing either wild-type AKAP12 or AKAP12?PKA and with ATR expression inhibited by siRNA. Cells were transfected with siRNA-resistant ATR-WT, ATR-S435A or ATR-S435D and treated with vehicle or forskolin (10 µM) and exposed to UVB (10 J/m2) or mock-treated. Data are expressed as the percent of chromatin-bound XPA to total cellular XPA. Treatments significantly different from vehicle were determined by one-way ANOVA; *P = 0.05. Data in all panels are representative from three independent experiments and error bars are standard deviation.

Fig 4: AKAP12 interacts with UV-induced photoproducts. (A and B) AKAP12 CRISPR-deleted HEK293 cells were transfected with wild-type AKAP12 or AKAP12?PKA were treated with either vehicle or forskolin (10 µM) and exposed to UVC (10 J/m2). The interaction between AKAP12 and CPDs at 30 min was determined by proximity ligation using anti-AKAP12 and anti-CPD antibodies. Green detection events signify juxtaposition between AKAP12 and [6-4]-PP in maximum intensity projection images. Nuclei were stained with DAPI (blue). Bar represents 50 µm. (C) Quantification of the AKAP12-CPD localization shown in panel A and B. At least 100 cells were counted from representative fields from two separate experiments. Values not sharing a common letter were significantly different as determined by one-way ANOVA; P = 0.05. (D and E) CRISPR-deleted AKAP12 HEK293 cells treated as described in (A). Interaction between AKAP12 and [6-4]-PP was determined by proximity ligation using anti-AKAP12 and anti-[6-4]-PP antibodies at 30 min after UV. Green detection events signify juxtaposition between AKAP12 and [6-4]-PP in maximum intensity projection images. Nuclei were stained with DAPI (blue). Bar represents 50 µm. (F) Quantification of the AKAP12-[6-4]-PP localization shown in panels d and e as described in (C). (G and H) Confocal imaging of AKAP12, XPA and ATR-pS435 in HEK293 cells transfected with wild-type AKAP12 or AKAP12?PKA. Cells were exposed to 10 J/m2 of UVC followed by in situ detergent extraction. Co-localization of AKAP12 (pink), XPA (green) and ATR-pS435 (red) is observed as white nuclear foci. Bar represents 50 µm. Data in all panels are representative from three independent experiments and error bars are standard deviation.

Fig 5: MC1R-cAMP signaling enhances interactions between XPA, ATR-pS435 and AKAP12 to a cisplatin-damaged substrate by ORiP. MC1R WT- or MC1R R151C-expressing HEK293 cells were pre-treated with either forskolin (10 µM), MSH (100 nM), MSH (100 nM) + ASIP (100 nM) or MSH (100 nM) + HBD3 (100 nM) 30 minutes before cisplatin treatment (100 µM) for 1 hr. Isolated nuclear extracts were incubated with a cisplatin-damaged DNA fragment (which acts as a substrate for NER) for a period of 30 minutes at 30 °C, as described in the Experimental Procedures. The interaction with the DNA substrate with (A) XPA, (B) ATR-pS435, (C) AKAP12 were determined using antibodies and quantified as described in Experimental Procedures for ORiP. Data are expressed as mean ± SEM from three independent experiments.