PCNA photo-crosslinking by diverse drugs (radiant light exposures 3.15 J cm⁻²). A, CV-1 cells were treated with 40 µM proflavine (PF), methylene blue (MB), acridine orange (AO), 9-aminoacridine (9AA) in serum-free medium, or with serum-free medium alone (C) for 30 min before irradiation. B, Proflavine (PF), m-AMSA (AMSA), doxorubicin (Dox), ethidium bromide (EtBr), camptothecin (CPT), ellipticine (EL), chloroquine (CH), sanguinarine (San), berberine (Ber), noscapine (Nos), and nitidine (Nit) were added to cells for 30 min before irradiation. C, Cells were exposed to different concentrations of hypericin or NPe6 for the indicated times before irradiation. Controls included untreated cells (0 µM) receiving the same light exposure, cells treated with 40 µM proflavine and light, cells treated with either 10 µM hypericin or 17 µM NPe6 for 1 hr but kept in the dark (dk), and cells treated with NPe6 for 1 hr in serum containing medium before irradiation (indicated with an asterisk). D, Cells were treated with proflavine or acridine orange at the indicated concentrations and then irradiated. Anti-PCNA Western blots (AO and light, upper left; PF and light, lower left) were visualized and quantitated with the BioRad ChemiDoc™ XRS imaging system. Quantitation of the 93 kDa PCNA (●) and the 154 kDa PCNA (○) bands is shown to the right of each Western blot.

The 154 kDa Band is a PCNA oligomer. A, Western blot of PCNA forms produced by 40 µM acridine orange and light (3.15 J cm⁻²) in HeLa cells. SDS PAGE (7% acrylamide) was done with the PCNA monomer run off the gel for maximum resolution in the high molecular weight range. Two molecular weight marker sets were used: EZ-Run™ pre-stained Rec Protein Ladder (left side markers) and Precision Plus Protein™ Dual color standards (right side markers). B, LC/MS/MS results. PCNA sequence coverage (bold) for the 154 kDa band determined by nano-LC/MS/MS of the Coomassie stained band after tryptic digestion, with sequence tags for the PCNA peptide NLAMGVNLTSMSK.

Photodynamic crosslinking of SV40 large T antigen and lamin B. A, SV40-transformed human fibroblasts (GM639) were briefly exposed to laboratory room lighting (1.3 W m⁻²) at bench top level for 5 min (0.039 J cm⁻²) and also exposed to more intense light (75 W m⁻²) in the irradiator (10 min, 4.5 J cm⁻²). SV40-infected CV-1 cells, with or without proflavine (40 µM, 30 min, 37 °C), were also exposed to light in the irradiator as indicated. SDS PAGE and Western blotting with anti-SV40 large T antigen (LT) were performed. B, Glutaraldehyde (GA) crosslinking of large T antigen in a RIPA extract, compared to large T antigen crosslinked by proflavine (40 µM) plus light (PF_L, 0.234 J cm⁻²) in GM639 cells. Large T antigen monomer was run off the 6% acrylamide SDS gel to achieve higher resolution of the high molecular weight crosslinked forms before Western blotting with anti-large T antigen antibody. C, GM639 cells were treated with 5 µM proflavine, 5 µM NPe6, and 5 µM hypericin for 1 hr at 37 °C, and then were irradiated (3.15 J cm⁻²). Anti-SV40 large T antigen Western blotting was done. Controls (C) were GM639 cells without drug or light exposure. D, GM639 cells were treated with hypericin (1 hr) or NPe6 (30 min) at 37°C and the indicated concentrations before irradiation (3.4 J cm⁻²). Western blotting with anti-lamin B antibody. Controls (C) were GM639 cells without drug or light exposure.

High molecular weight PCNA forms produced by proflavine and light. A. CV-1 cells in SFM were exposed to room lighting for 5 min on the bench top (0.039 J cm⁻²) without (C_RL), or with (PF_RL) 10 µM proflavine treatment (37°C, 30 min). Other cells were UV-irradiated (UV, 30 J m⁻²), incubated 3hr in serum-containing MEM, and Western blotted with PCNA antibody (PC-10). MW, Molecular weight; PCNA, PCNA monomer; Ub-PCNA, monoubiquitinated PCNA; 93 kDA, 93 kilodalton PCNA form. B, CV-1, MCF-7, and HeLa cells in SFM were exposed to light in the irradiator (7 min, 75 W m⁻², 3.15 J cm⁻²) without (C_L) or with 40 µM proflavine (PF_L). C, A RIPA lysate of MCF-7 cells was treated with glutaraldehyde (GA, 0.025%, 10 min, 37°C), glycine was added to 0.15 M to stop protein crosslinking, and the sample was Western blotted (anti-PCNA). Minor high molecular weight PCNA bands are indicated (Minor Bands) with 93 kDa and 154 kDa bands. D, HeLa cells and HeLa cells expressing HA-tagged PCNA (HA-PCNA HeLa) in SFM were exposed to light (3.15 J cm⁻²) without (C_L) or with 40 µM proflavine (PF_L), then Western blotted with HA tag antibody. Asterisk: non-specific band. HeLa cells expressing HA tagged PCNA were also immunoprecipitated with HA-tag antibody, then Western blotted with PCNA antibody. E, CV-1 cells treated with 40 µM proflavine in SFM were irradiated for different times (75 W m⁻²), and anti-PCNA Western blotting was performed. The zero time point is a dark control. F, CV-1 cells treated with the E1 ubiquitin ligase inhibitor, PYR-41 (25 µM), proflavine (40 µM) and light (3.15 J cm⁻²), were analyzed by PCNA Western blotting. ts85 cells, capable of ubiquitination at 32°C but not at 39°C, were incubated at each temperature for 16 hr, then treated with 40 µM proflavine and light (3.15 J cm⁻²) and analysed by PCNA Western blotting. G, CV-1 cells were treated with 40 µM acridine orange and light (3.15 J cm⁻²). SDS lysates were immunoprecipitated with anti-PCNA antibody, and Western blotted with anti-ubiquitin antibody (left) before re-probing with anti-PCNA antibody (right).

The 93 kDa band is the crosslinked PCNA trimer. A, HeLa cells were treated with 40 µM proflavine (PF) and light (3.15 J cm⁻²) or with 1.5 % formaldehyde (HCHO) for 30 min at room temperature. The proflavine plus light treated cells were lysed with SDS lysis buffer, and the formaldehyde treated cells were lysed with RIPA buffer following the protocol of Naryzhny et al. [13, 37]. The proteins from the two samples were Western blotted with PC10 antibody. A longer film exposure of the HCHO lane shows the 154 kDa PCNA positive band. B, PCNA from cells exposed to 40 µM proflavine and light (PF, 3.15 J cm⁻²) and control cells exposed to light alone (C) was immunoprecipitated from a HeLa cell RIPA lysate with PC10 antibody. Proteins of the immunoprecipitate were visualized by Coomassie staining. C, PC10 immunoprecipitated HeLa samples were Western blotted with PC10 and C20 antibodies. The IgG heavy chain from the immunoprecipitation is indicated (IgG HC). D, The 93 kDa Coomassie stained band was excised, trypsin digested, and processed for LC/MS/MS. PCNA amino acid sequences detected by LC/MS/MS are indicated in bold. Sequence tags for the unique PCNA tryptic peptide AEDNADTLALVFEAPNQEK are shown.

Tests for ROS causing PCNA crosslinking. CV-1 cells were lysed in SDS lysis buffer after the treatments, and the lysates were subjected to SDS gel electrophoresis (10% acrylamide). Western blotting was done with PC10 antibody against PCNA. A, Hydrogen peroxide exposure. Cells were treated with 5 mM H₂O₂ in SFM for 30 min, then the H₂O₂ medium was replaced with MEM supplemented with 10% calf serum for the times indicated. Untreated cells were a control (C). For the UV control, cells were exposed to germicidal UV light (30 J m⁻²), then extracted 3 hr later. Cells were treated with 40 or 250 µM paraquat (30 min, 37°C), or 40 µM proflavine and light (0.2 J cm⁻²) as a control. B, Oxygen dependence. Cells were changed to SFM, with or without 40 µM proflavine, flushed with room air (Air) or with nitrogen (N₂) for 1 hr, then irradiated (1.35 J cm⁻²). Controls include cells not treated with proflavine (C), and cells treated with proflavine and flushed with air, but kept in the dark (Dark). C, Histidine suppression. Left: Cells were pretreated with SFM or with SFM containing 50 mM L-histidine (Hist) for 1.5 hr, and with 40 µM proflavine, methylene blue or acridine orange for the last 30 min. After incubation in the dyes, the cells were irradiated (0.9 J cm⁻²). Right: Cells were pretreated with SFM or with 100 mM histidine in SFM for 1.5 hr and then with camptothecin (200 µM), doxorubicin (200 µM), NPe6 (1.7 µM), or hypericin (0.5 µM) before irradiation (3.15 J cm⁻²). D, D₂O enhancement. Left: Cells were treated for the times indicated with SFM made up in 99.9% D₂O or in normal SFM, then the cells were irradiated (3.15 J cm⁻²) either in D₂O alone or in D₂O containing 40 µM PF. Light irradiated control (C_L) and proflavine (PF_L, 40 µM) experiments were in media made up with deionized water. Right: Cells were treated with D₂O media for 3 hr, then with camptothecin (200 µM), doxorubicin (200 µM), NPe6 (1.7 µM), or hypericin (0.5 µM) before irradiation (3.15 J cm⁻²). Controls were identically treated but with SFM made in deionized water. Irradiated control cells (C_L) are indicated.