Fig 2: Expression of E1 during the first 36 hours after infection with HAdV5.Arrested IMR-90 cells were infected with dl309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: E1A = 0.001; E1B-19k < 0.0001; E1B-55k < 0.0001. All time-points between 17 and 36 hours were statistically significant.

Fig 3: Expression of VAII RNA during the first 36 hours after infection with HAdV5.Arrested IMR-90 cells were infected with dl309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the p = 0.0027. All time-points between 17 and 36 hours were statistically significant.

Fig 4: HULLK expression in prostate cancer. a Expression of HULLK in a panel of PCa and normal prostate epithelial cell lines cultured in complete growth media. qPCR was performed with two independent LCK primer sets targeting exon 2 (E2) and the 3’UTR (3 U), n = 3. b HULLK expression in C4–2, MCF7, BT549, MDA-MB-231, HeLa, and PANC1 cell lines grown in complete growth media. qPCR was performed with LCK primers targeting the 3’UTR, n = 3. c A significant positive correlation between HULLK expression and PCa grade. 33 FFPE tissue samples were obtained from the Biorepository and Tissue Research Facility at the University of Virginia. These samples represent biopsies and surgical resections collected in 2016 from PCa patients presenting with Gleason Score 6–10. Normal and cancerous regions were demarcated and 1.5 mm punches were collected. ddPCR was performed on the QX200 droplet digital PCR system (Bio-Rad). d 16 fresh-frozen tissue samples from Gleason Score 6–10 PCa patients were acquired from Dr. Ganesh Raj from the University of Texas Southwestern Medical Center. These samples were surgically resected from May to December in 2017, placed in RNALater (ThermoFisher), and frozen immediately. e Statistical analysis of the association between the LCK E13 to LCK E4 ratio and grade was performed using linear regression models, following transformation to the log scale. The analyses were conducted separately for frozen and FFPE samples, with a test for interaction used to test whether the association between the E13 to E4 ratio and grade was the same in frozen samples as in FFPE samples. f HULLK expression in normal and tumor and g low Gleason [scores 6 and 7(3 + 4)] and high Gleason score [scores 7(4 + 3), and 8–10] in the PRAD TCGA cohort

Fig 5: Expression of E2, E3, and E4 during the first 36 hours after infection with HAdV5.Arrested IMR-90 cells were infected with dl309 at an MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: DBP < 0.001; DNA pol = 0.0058; E3 = 0.0086; E4 orf3 < 0.0001; E4 orf6/7 < 0.0001. All time-points between 17 and 36 hours were statistically significant.