Fig. 7: SERPINB3 is sufficient to protect cervical cancer cells against radiation-induced death, but requires a functional reactive site loop.

a Western blot of SiHa and C33A parental cervical tumor cell lines (P) which do not express SERPINB3 protein at baseline, and stable clones engineered to express wild-type SERPINB3 (B3), SERPINB3-A341R mutant (A341R), or empty vector control (VC). b Clonogenic survival of SiHa cells expressing VC, B3 or B3-A341R constructs, following increasing doses of RT, individual data points with fit linear quadratic curve is shown. Dose modifying factor (DMF) displayed with reference to VC. c Relative proliferation of C33A cells expressing VC, B3 or B3-A341R treated with RT normalized to Sham-treated cells at 24 h post-treatment, and measured by relative light units (RLU) of CellTiter-Glo luminescent reagent measuring cellular ATP. Individual data points (n = 6) are shown mean indicated by bar height and standard deviations are plotted. P-value for one-way ANOVA shown. d, e Mean tumor volume curves of flank xenograft generated from C33A cells expressing VC, B3-wt or B3-A341R treated with sham (d) or 10 Gy tumor-directed radiation (e) with tumors harvested at indicated time post-treatment. f Time to tumor doubling in days for each of the tumors. Individual data points are shown, tumors not doubling by the termination of the experiment were included as a data point on the last day of the experiment. P-value for paired t-test between sham and 10 Gy groups for each tumor type are shown. g Harvested tumors from sham (left) or 10 Gy (right) irradiated tumors from experiment shown above. h Fragments per kilobase of transcript per million mapped reads (FPKM) of cysteine cathepsin protease gene transcripts in 68 patient specimens. CTSB and CTSH have predominantly exopeptidase activity (exo>endo), CTSC and CTSX have exclusively exopeptidase activity (exo), and the remainder have exclusively endopeptidase activity (endo). Inset shows the same data zoomed to 0–500 FPKM. i Western blot of SW756 cells with wild-type or knock SERPINB3 (B3-WT and B3-KO, respectively) and wild-type or knockout cathepsin L (CTSL-WT and CSTL-KO respectively) Arrows indicate pro-, single chain, and mature forms of cathepsin enzymes. j Representative histogram of percent Sytox + cells 96 h post-treatment with sham or 30 Gy in SW756-B3-WT/KO–CSTL-WT/KO backgrounds. Multiple comparisons from one-way ANOVA is shown for indicated comparisons (ns non-significant, ** = p-value < 0.01, **** = p-value < 0.0001). Individual data points with bar height at the mean % Sytox-positive quantified in 2–4 fields of view of triplicate well (n = 3 per condition), and is a representative one of three biologic replicate experiments.