Steady state levels of Casp6, Casp1, and Casp3 in human adult tissues.

<p><b>A</b>. Representative western blot (series #2) of 100 µg of total proteins from adult tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody. <b>B</b>. Age of adult tissues used for the study. <b>C</b>. Bar graph of the average of proCasp6 levels relative to that in colon arbitrarily placed at 100. Data represent the individual values as well as mean and SEM of 3 independent experiments. <b>D</b>. Coomassie stained gel of proteins from tissues in series 2.</p

Micrographs of immunohistochemical analyses of adult normal and cancerous colon with 1277 neoepitope antisera against the active p20 subunit of Casp6.

<p>Normal colonic mucosa (A) and crypt openings (B), colonic adenocarcinomas (C) and crypt openings (D) immunostained with anti-p20Casp6 neoepitope antisera. E. Larger magnification of normal colon showing active Casp6-positive epithelial cell sloughing off in the lumen. F. Western blot of 100 µg of total proteins from normal and cancerous colon tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody.</p

Steady state levels of Casp6, Casp1, and Casp3 in 8 different human fetal stomachs.

<p>Representative western blots containing 100 µg total protein/lane from fetal stomachs of different developmental ages with <b>A</b>. Neomarker anti-Casp6 antisera (top panel), neoepitope 10630 antisera (second panel), and β-Actin antibody (third panel), <b>B. Micrograph of human fetal stomach stained with 10630 anti-active Casp6 antiserum. C& D.</b> Western blot of (<b>C</b>) anti-Casp1 antisera (ProCasp1), neoepitope antisera to the p20 subunit of Casp1 (p20Casp1) and β-Actin antibody, and (<b>D</b>) anti-Casp3 antisera (ProCasp3), neoepitope antisera to the p20 subunit of Casp3 (p20Casp3) and β-Actin antibody.</p

Casp6 levels are increased in AOM/DSS-induced colon tumors.

<p>(A) Schematic overview of the inflammation-induced cancer model. Age and sex-matched mice were injected with Azoxymethane (AOM) intraperitoneally at a dose of 6 mg/kg body weight. After one week, mice were treated with 2% DSS in the drinking water for 5 days, then followed by 16 days of regular water. This cycle was repeated once. Mice were sacrificed wither 12 or 20 weeks post AOM injection. (B) Casp6 mRNA was assessed by qRT-PCR on isolated RNA from tumors (T) and adjacent normal appearing tissues (N). HPRT was used as a loading control (n = 5). Statistical significance was assessed with a paired t-test. * p<0.05. (C) Casp6 protein expression in colon tissue extracts of WT mice was measured by western blot analysis with actin as a loading control (n = 3). (D) Specific VEIDase Casp6 activity in extracts from WT colons (n = 3). Statistical significance was assessed with a paired t-test. ** p<0.01. (E) Cleavage of lamin A by activated Casp6 was also tested using anti-lamin AΔCasp6 antibody and anti-lamin A+C used as equal loading control in tumors and normal appearing tissue from WT and KO Casp6 mice.</p

Micrographs of immunohistochemical analyses of fetal tissues with 1277 neoepitope antisera detecting the active p20 subunit of Casp6.

<p>A. At low power (original magnification, 100x), immunopositive cells (arrows) in the dorsal root ganglion (DRG), in the anterior horn (AH), and in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). B. High power view of dorsal root ganglion (DRG: original magnification, 400×) indicating mitotic activity (solid arrowhead), features of early neuronal differentiation (open arrowheads) and several apoptotic immunopositive cells (arrows). C. High power view of anterior horn (AH: original magnification, 400×) indicating dividing cells (solid arrow heads), early maturation of cells (open arrowheads), and an immunopositive apoptotic precursor cell (arrow). WM; white matter). D. High power view of dorsal spinal cord (original magnification, 400×). Immunopositive finely granular (synaptic) pattern in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). DRG, dorsal root ganglion; DR, dorsal root; PH, posterior horn). E. Extensive extramedullary hematopoiesis (EMH) in the sinusoids that separate the cords of hepatocytes (HC) in the liver (original magnification 400×). In the hematopoietic islands, there are many immunopositive apoptotic cells (arrows).</p

Susceptibility of Casp6 overexpressing mice to AOM-DSS treatment.

<p>(<b>A</b>) RT-PCR of human Casp6 and Cre recombinase in colons of KI and Cre mice. (B) Western blot of human Casp6 protein and active subunits in mice colon proteins. Cleavage of lamin A by activated Casp6 was also tested using anti-lamin AΔCasp6 antibody and anti-lamin A+C used as equal loading control. (C) Immunohistochemistry of WT/WT, KI/WT, WT/Cre and KI/Cre mice colons with anti-p20 active Casp6 neoepitope antisera. (D) Percent weight change measured weekly during AOM-DSS treatment. The black boxes represent the period of DSS treatment. (E) Human Casp6 mRNA levels were assessed by qRT-PCR in tumors and adjacent normal appearing tissue from WT/WT, WT/Cre, KI/WT and KI/Cre mice colons. HPRT was used as a loading control (n = 3). One-way ANOVA followed by Tukey-Kramer post hoc analysis was performed to determine statistical significance. * p<0.05, ** p<0.01 comparing with Normal KI/Cre and ### p<0.001 comparing with Tumor KI/Cre. (F) Specific VEIDase Casp6 activity for extracts from WT/WT, WT/Cre, KI/WT and KI/Cre colons (n = 3). One-way ANOVA followed by Tukey-Kramer post hoc analysis was performed to determine statistical significance. * p<0.05 compares normal versus tumor tissues and # p<0.05 compares with tumors from KI/Cre. (G) Western blot analyses for pro-Casp6 and active p20 subunit of Casp6 in colon protein extracts from WT/WT, WT/Cre, KI/WT and KI/Cre mice. (H) Number of hyperplasia per colon induced by AOM-DSS treatment 12 weeks after AOM injection. (I) Number of tumors per colon induced by AOM-DSS treatment 12 weeks after AOM injection. (J) Tumor load per mouse in mice after AOM/DSS treatment. (K) Number of tumors per mouse located in proximal, middle or distal part of colons. (H-K) Statistics were performed using one way ANOVA followed by Tukey-Kramer post hoc analysis.</p

Histological grades of AOM/DSS-induced colon tumors in KI/Cre (A) and KO (B) mice.

<p>Abbreviations: LGD, adenoma with low-grade dysplasia; HGD, adenoma with high-grade dysplasia.</p><p>Histological grades of AOM/DSS-induced colon tumors in KI/Cre (A) and KO (B) mice.</p

Tumor cell proliferation and apoptosis in Casp6 overexpressing and deficient mice.

<p>(A) Representative Ki67 immunohistochemistry in tumors in WT/WT, WT/Cre, KI/WT and KI/Cre mice given AOM/DSS (scale bar, 100 µm). (B) Number of Ki67 positive cells in colonic tumors from mice (N = 3). Data expressed as means +/− SEM. (C) Representative Ki67 immunohistochemistry in tumors from WT/WT, WT/KO, and KO/KO mice given AOM/DSS (scale bar, 100 µm). (D) Number of Ki67 positive cells in colonic tumors from KO mice (N = 3). Data expressed as means +/− SEM. (E) Representative Cleaved Casp3 immunohistochemistry in positive control (fetal ischemic brain), negative control (colon without Primary Antibody) and a colon from AOM/DSS treated mouse (scale bar, 50 µm). Number of cleaved Casp3 positive cells in colonic tumors from mice (N = 3). Data expressed as means +/− SEM. (F) Number of Cleaved Casp3 positive cells in colonic tumors from KO mice (N = 3). Data expressed as means +/− SEM.</p

Susceptibility of Casp6 deficient mice to AOM/DSS treatment.

<p>(A) Percent weight change measured weekly during AOM-DSS treatment. The black boxes represent the period for DSS treatment. Inset: Casp6 mRNA was assessed by RT-PCR with primers amplifying full length Casp6 mRNA in WT and truncated Casp6 mRNA in the KO. (B) Number of hyperplasia per colon induced by AOM-DSS treatment 20 weeks post AOM injection. (C) Number of tumors per colon induced by AOM-DSS treatment 20 weeks after AOM injection. (D) Tumor load per mouse in mice after AOM/DSS treatment. (E) Number of tumors per mouse located in proximal, middle or distal part of colons. (B-E) Statistics were performed using one way ANOVA followed by Tukey-Kramer post hoc analysis.</p

Hyperplasia and Tumor (macroscopic) incidence (%) in KI/Cre (A) and KO (B) mice treated with AOM/DSS.

<p>Data on the hyperplasia and tumors incidence were analyzed by X² test. Differences were considered significant if the probability of the difference by chance was less than 5 in 100 (P<0.05).</p><p>Hyperplasia and Tumor (macroscopic) incidence (%) in KI/Cre (A) and KO (B) mice treated with AOM/DSS.</p