Exp. inhibitor of glucosylceramide synthase, the enzyme in charge of catalyzing the glycosylation of ceramides to create glucosylceramides. Inhibition of glucosylceramide synthase attenuated the build up from the hexosylceramides and exacerbated ceramide build up in the renal cortex pursuing treatment of mice with cisplatin. Raising ceramides and reducing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI relating to markers of kidney function, kidney damage, inflammation, cell tension, and apoptosis. Under circumstances of high ceramide era, data claim that rate of metabolism of ceramides to glucosylceramides buffers kidney ceramides and assists attenuate kidney damage.Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Clear, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced severe kidney damage. as used and promulgated by the united states Country wide Institutes of Health insurance and were authorized by the Institutional Pet Care and Make use of Committee. At 24 h post-glycerol shot, at the maximum of damage (43), the kidneys had been used for planning of homogenate for dimension of sphingolipids and enzyme actions as referred to below. Dimension of kidney function BUN (40146) and SCr (C7548-120) amounts were established on serum examples using products from AMS Diagnostics (Weston, FL) and Stage Scientific Inc. (Canton, MI), respectively, following a manufactures instructions. Proteins quantification and Traditional western blot evaluation Kidney cortex was homogenized in cell removal buffer (Thermo Fisher Scientific, Waltham, MA), including an entire Protease Inhibitor Cocktail tablet and a Phosphatase Inhibitor Cocktail tablet (Roche, Indianapolis, IN). Homogenates had been centrifuged at 15,000 for 10 min at 4C. Supernatants had been removed, combined, aliquoted, LW6 (CAY10585) and kept at ?80C. Proteins concentrations were established using Bradford reagent (Bio-Rad, Hercules, CA). Kidney homogenate (40 g) was separated on 4C12% gradient Tris-glycine-SDS polyacrylamide gels and used in PVDF membranes LW6 (CAY10585) and clogged in 5% (w/v) dried out dairy in TBS with 0.1% Tween 20 (TBST) for 1 h. Membranes were incubated with major antibody in 4C overnight. Membranes were after that washed 3 x for 5 min each with TBST including 5% (w/v) dried out dairy. After incubation for 1 h at area temperature with supplementary antibodies conjugated with HRP (1:10,000) in TBST filled with 1.25% (w/v) dried milk, membrane protein were detected by chemiluminescence substrate. Antibodies had been bought from Cell Signaling (Beverly, MA) unless in any other case observed: inositol needing enzyme-1 (IRE1) (#3294), ERK (#4695), phosphorylated (p)- ERK (#4370), cleaved caspase 3 (#9664), cleaved caspase 8 (#8592), C/EBP homologous proteins (CHOP) (#2895), c-Jun N-terminal kinase (JNK) (#9258), phosphorylated-c-Jun N-terminal kinase (p-JNK) (#4668), proliferating cell nuclear antigen (PCNA) (#13110), and -actin (catalog amount A2228; Sigma Aldrich). Gene appearance RNA was isolated using TRIzol (Thermo Fisher Scientific) per the producers process. cDNA was synthesized with high-capacity cDNA change transcriptase PCR (Thermo Fisher Scientific) per the producers guidelines. TNF- (Mm00443258_m1), interleukin (IL)-6 (Mm00446190_m1), chemokine (C-X-C theme) ligand 1 (CXCL1) (Mm04207460_m1), kidney damage molecule-1 (KIM-1) (Mm00506686), as well as the housekeeping gene, (LTA) being a proximal tubule marker. Quickly, kidney areas (5 m) had been deparaffinized and rehydrated, antigens had been unmasked, accompanied by preventing of endogenous peroxidases. Areas were further obstructed with 5% goat serum with 0.1% Triton X-100 in PBS, accompanied by incubation with HRP-conjugated LTA (1:500) for 2 h at area temperature. HRP-LTA was discovered with Nova Crimson peroxidase (SK-4800; Vector Labs, Burlingame, CA). Next, areas had been obstructed for biotin LW6 (CAY10585) and avidin, accompanied by anti-PCNA (1:16,000) incubation right away at 4C. Areas were after that incubated with biotinylated goat anti-rabbit IgG antibody (1:25,000, BA-1000; Vector Laboratories) for 30 min at area heat range. Vector ABC reagent (PK-7100; Vector Laboratories) was put into each section and incubated for 30 min at area heat range. PCNA was after that visualized with Nova Crimson DAB substrate (SK-4800; Vector Laboratories). Slides had been counterstained with improved Mayers hematoxylin (72804; Thermo Fisher Scientific), and dehydrated accompanied by installation with Permount (SP15; Thermo Fisher Scientific). Pictures were visualized utilizing a Nikon Eclipse E600 microscope (Nikon Company, Tokyo, Japan). For quantification of PCNA, positive cells that stained dark brown in color had been enumerated and summed per square micron (200 magnification field). TUNEL assays had been performed on paraffin inserted tissue areas using Apoptag Crimson in situ apoptosis recognition package per the producers guidelines (S7165; Millipore,.R., and Siskind L. glucosylceramide synthase, the enzyme in charge of catalyzing the glycosylation of ceramides to create glucosylceramides. Inhibition of glucosylceramide synthase attenuated the deposition from the hexosylceramides and exacerbated ceramide deposition in the renal cortex pursuing treatment of mice with cisplatin. Raising ceramides and lowering glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI regarding to markers of kidney function, kidney damage, inflammation, cell tension, and apoptosis. Under circumstances of high ceramide era, data claim that fat burning capacity of ceramides to glucosylceramides buffers kidney ceramides and assists attenuate kidney damage.Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Clear, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced severe kidney damage. as followed and promulgated by the united states Country wide Institutes of Health insurance and were accepted by the Institutional Pet Care and Make use of Committee. At 24 h post-glycerol shot, at the top of damage (43), the kidneys had been used for planning of homogenate for dimension of sphingolipids and enzyme actions as defined below. Dimension of kidney function BUN (40146) and SCr (C7548-120) amounts were driven on serum examples using sets from AMS Diagnostics (Weston, FL) and Stage Scientific Inc. (Canton, MI), respectively, following manufactures instructions. Proteins quantification and Traditional western blot evaluation Kidney cortex was homogenized in cell removal buffer (Thermo Fisher Scientific, Waltham, MA), filled with an entire Protease Inhibitor Cocktail tablet and a Phosphatase Inhibitor Cocktail tablet (Roche, Indianapolis, IN). Homogenates had been centrifuged at 15,000 for 10 min at 4C. Supernatants had been removed, blended, aliquoted, and kept at ?80C. Proteins concentrations were driven using Bradford reagent (Bio-Rad, Hercules, CA). Kidney homogenate (40 g) was separated on 4C12% gradient Tris-glycine-SDS polyacrylamide gels and used in PVDF membranes and obstructed in 5% (w/v) dried out dairy in TBS with 0.1% Tween 20 (TBST) for 1 h. Membranes had been incubated with principal antibody right away at 4C. Membranes had been then washed 3 x for 5 min each with TBST filled with 5% (w/v) dried out dairy. After incubation for 1 h at area temperature with supplementary antibodies conjugated with HRP (1:10,000) in TBST filled with 1.25% (w/v) dried milk, membrane protein were detected by chemiluminescence substrate. Antibodies had been bought from Cell Signaling (Beverly, MA) unless in any other case observed: inositol needing enzyme-1 (IRE1) (#3294), ERK (#4695), phosphorylated (p)- ERK (#4370), cleaved caspase 3 (#9664), cleaved caspase 8 (#8592), C/EBP homologous proteins (CHOP) (#2895), c-Jun N-terminal kinase (JNK) (#9258), phosphorylated-c-Jun N-terminal kinase (p-JNK) (#4668), proliferating cell nuclear antigen (PCNA) (#13110), and -actin (catalog amount A2228; Sigma Aldrich). Gene appearance RNA was isolated using TRIzol (Thermo Fisher Scientific) per the producers process. cDNA was synthesized with high-capacity cDNA change transcriptase PCR (Thermo Fisher Scientific) per the producers guidelines. TNF- (Mm00443258_m1), interleukin (IL)-6 (Mm00446190_m1), chemokine (C-X-C theme) ligand 1 (CXCL1) (Mm04207460_m1), kidney damage molecule-1 (KIM-1) (Mm00506686), as well as the housekeeping gene, (LTA) being a proximal tubule marker. Quickly, kidney areas (5 m) had been deparaffinized and rehydrated, antigens had been unmasked, accompanied by preventing of endogenous peroxidases. Areas were further obstructed with 5% goat serum with 0.1% Triton X-100 in PBS, accompanied by incubation with HRP-conjugated LTA (1:500) for 2 h at area temperature. HRP-LTA was discovered with Nova Crimson peroxidase (SK-4800; Vector Labs, Burlingame, CA). Next, areas were obstructed for avidin and biotin, accompanied by anti-PCNA (1:16,000) incubation right away at 4C. Areas were after that incubated with biotinylated goat anti-rabbit IgG antibody (1:25,000, BA-1000; Vector Laboratories) for 30 min at area heat range. Vector ABC reagent (PK-7100; Vector Laboratories) was put into each section and incubated for 30 min at area heat range. PCNA was after that visualized with Nova Crimson DAB substrate (SK-4800; Vector Laboratories). Slides had been counterstained with improved Mayers hematoxylin (72804; Thermo Fisher Scientific), and dehydrated accompanied by installation with Permount (SP15; Thermo Fisher Scientific). Pictures were visualized utilizing a Nikon Eclipse E600 microscope (Nikon Company, Tokyo, Japan). For quantification of PCNA, positive cells that stained dark brown in color had been enumerated and summed per square micron (200 magnification field). TUNEL assays had been performed on paraffin inserted tissue areas using Apoptag Crimson in situ apoptosis recognition package per the producers guidelines (S7165; Millipore, Temecula, CA)..PCNA-positive cells are reddish dark brown in color, while LTA-positive tubules are dark. mice with cisplatin. Raising ceramides and lowering glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI regarding to markers of kidney function, kidney damage, inflammation, cell tension, and apoptosis. Under circumstances of high ceramide era, data claim that fat burning capacity of ceramides to glucosylceramides buffers kidney ceramides and assists attenuate kidney damage.Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Clear, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced severe kidney damage. as followed and promulgated by the united states Country wide Institutes of Health insurance and were accepted by the Institutional Pet Care and Make use of Committee. At 24 h post-glycerol shot, at the top of damage (43), the kidneys had been used for planning of homogenate for dimension of sphingolipids and enzyme actions as referred to below. Dimension of kidney function BUN (40146) and SCr (C7548-120) amounts were motivated on serum examples using products from AMS Diagnostics (Weston, FL) and Stage Scientific Inc. (Canton, MI), respectively, following manufactures instructions. Proteins quantification and Traditional western blot evaluation Kidney cortex was homogenized in cell removal buffer (Thermo Fisher Scientific, Waltham, MA), formulated with an entire Protease Inhibitor Cocktail tablet and a Phosphatase Inhibitor Cocktail tablet (Roche, Indianapolis, IN). Homogenates had been centrifuged at 15,000 for 10 min at 4C. Supernatants had been removed, blended, aliquoted, and kept at ?80C. Proteins concentrations were motivated using Bradford reagent (Bio-Rad, Hercules, CA). Kidney COG3 homogenate (40 g) was separated on 4C12% gradient Tris-glycine-SDS polyacrylamide gels and used in PVDF membranes and obstructed in 5% (w/v) dried out dairy in TBS with 0.1% Tween 20 (TBST) for 1 h. Membranes had been incubated with major antibody right away at 4C. Membranes had been then washed 3 x for 5 min each with TBST formulated with 5% (w/v) dried out dairy. After incubation for 1 h at area temperature with supplementary antibodies conjugated with HRP (1:10,000) in TBST formulated with 1.25% (w/v) dried milk, membrane protein were detected by chemiluminescence substrate. Antibodies had been bought from Cell Signaling (Beverly, MA) unless in any other case observed: inositol needing enzyme-1 (IRE1) (#3294), ERK (#4695), phosphorylated (p)- ERK (#4370), cleaved caspase 3 (#9664), cleaved caspase 8 (#8592), C/EBP homologous proteins (CHOP) (#2895), c-Jun N-terminal kinase (JNK) (#9258), phosphorylated-c-Jun N-terminal kinase (p-JNK) (#4668), proliferating cell nuclear antigen (PCNA) (#13110), and -actin (catalog amount A2228; Sigma Aldrich). Gene appearance RNA was isolated using TRIzol (Thermo Fisher Scientific) per the producers process. cDNA was synthesized with high-capacity cDNA change transcriptase PCR (Thermo Fisher Scientific) per the producers guidelines. TNF- (Mm00443258_m1), interleukin (IL)-6 (Mm00446190_m1), chemokine (C-X-C theme) ligand 1 (CXCL1) (Mm04207460_m1), kidney damage molecule-1 (KIM-1) (Mm00506686), as well as the housekeeping gene, (LTA) being a proximal tubule marker. Quickly, kidney areas (5 m) had been deparaffinized and rehydrated, antigens had been unmasked, accompanied by preventing of endogenous peroxidases. Areas were further obstructed with 5% goat serum with 0.1% Triton X-100 in PBS, accompanied by incubation with HRP-conjugated LTA (1:500) for 2 h at area temperature. HRP-LTA was discovered with Nova Crimson peroxidase (SK-4800; Vector Labs, Burlingame, CA). Next, areas were obstructed for avidin and biotin, accompanied by anti-PCNA (1:16,000) incubation over night at 4C. Areas were after that incubated with biotinylated goat anti-rabbit IgG antibody (1:25,000, BA-1000; Vector Laboratories) for 30 min at area temperatures. Vector ABC reagent (PK-7100; Vector Laboratories) was put into each section and incubated for 30 min at area temperatures. PCNA was after that visualized with Nova Crimson DAB substrate (SK-4800; Vector Laboratories). Slides had been counterstained with customized Mayers hematoxylin (72804; Thermo Fisher Scientific), and dehydrated accompanied by installation with Permount (SP15; Thermo Fisher Scientific). Pictures had been visualized using.J. the deposition from the hexosylceramides and exacerbated ceramide deposition in the renal cortex pursuing treatment of mice with cisplatin. Raising ceramides and lowering glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI regarding to markers of kidney function, kidney damage, inflammation, cell tension, and apoptosis. Under circumstances of high ceramide era, data claim that fat burning capacity of ceramides to glucosylceramides buffers kidney ceramides and assists attenuate kidney damage.Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Clear, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced severe kidney damage. as followed and promulgated by the united states Country wide Institutes of Health insurance and were accepted by the Institutional Pet Care and Make use of Committee. At 24 h post-glycerol shot, at the top of damage (43), the kidneys had been used for preparation of homogenate for measurement of sphingolipids and enzyme activities as described below. Measurement of kidney function BUN (40146) and SCr (C7548-120) levels were determined on serum samples using kits from AMS Diagnostics (Weston, FL) and Point Scientific Inc. (Canton, MI), respectively, following the manufactures instructions. Protein quantification and Western blot analysis Kidney cortex was homogenized in cell extraction buffer (Thermo Fisher Scientific, Waltham, MA), containing a Complete Protease Inhibitor Cocktail tablet and a Phosphatase Inhibitor Cocktail tablet (Roche, Indianapolis, IN). Homogenates were centrifuged at 15,000 for 10 min at 4C. Supernatants were removed, mixed, aliquoted, and stored at ?80C. Protein concentrations were determined using Bradford reagent (Bio-Rad, Hercules, CA). Kidney homogenate (40 g) was separated on 4C12% gradient Tris-glycine-SDS polyacrylamide gels and transferred to PVDF membranes and then blocked in 5% (w/v) dried milk in TBS with 0.1% Tween 20 (TBST) for 1 h. Membranes were incubated with primary antibody overnight at 4C. Membranes were then washed three times for 5 min each with TBST containing 5% (w/v) dried milk. After incubation for 1 h at room temperature with secondary antibodies conjugated with HRP (1:10,000) in TBST containing 1.25% (w/v) dried milk, membrane proteins were detected by chemiluminescence substrate. Antibodies were purchased from Cell Signaling (Beverly, MA) unless otherwise noted: inositol requiring enzyme-1 (IRE1) (#3294), ERK (#4695), phosphorylated (p)- ERK (#4370), cleaved caspase 3 (#9664), cleaved caspase 8 (#8592), C/EBP homologous protein (CHOP) (#2895), c-Jun N-terminal kinase (JNK) (#9258), phosphorylated-c-Jun N-terminal kinase (p-JNK) (#4668), proliferating cell nuclear antigen (PCNA) (#13110), and -actin (catalog number A2228; Sigma Aldrich). Gene expression RNA was isolated using TRIzol (Thermo Fisher Scientific) per the manufacturers protocol. cDNA was synthesized with high-capacity cDNA reverse transcriptase PCR (Thermo Fisher Scientific) per the manufacturers instructions. TNF- (Mm00443258_m1), interleukin (IL)-6 (Mm00446190_m1), chemokine (C-X-C motif) ligand 1 (CXCL1) (Mm04207460_m1), kidney injury molecule-1 (KIM-1) (Mm00506686), and the housekeeping gene, (LTA) as a proximal tubule marker. Briefly, kidney sections (5 m) were deparaffinized and rehydrated, antigens were unmasked, followed by blocking of endogenous peroxidases. Sections were further blocked with 5% goat serum with 0.1% Triton X-100 in PBS, followed by incubation with HRP-conjugated LTA (1:500) for 2 h at room temperature. HRP-LTA was detected with Nova Red peroxidase (SK-4800; Vector Labs, Burlingame, CA). Next, sections were blocked for avidin and biotin, followed by anti-PCNA (1:16,000) incubation overnight at 4C. Sections were then incubated with biotinylated goat anti-rabbit IgG antibody (1:25,000, BA-1000; Vector Laboratories) for 30 min at room temperature. Vector ABC reagent (PK-7100; Vector Laboratories) was added to each section and incubated for 30 min at room temperature. PCNA was then visualized with Nova Red DAB substrate (SK-4800; Vector Laboratories). Slides were counterstained with modified Mayers hematoxylin (72804; Thermo Fisher Scientific), and then dehydrated followed by mounting with Permount (SP15; Thermo Fisher Scientific). Images were visualized using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan). For quantification of PCNA, positive cells that stained brown in color were enumerated and then summed per square micron (200 magnification field). TUNEL assays were performed on paraffin embedded tissue sections using Apoptag Red in situ apoptosis detection kit per the manufacturers instructions (S7165; Millipore, Temecula, CA). Sections were counterstained with DAPI and mounted with VECTASHIELD antifade mounting medium. Slides were visualized via immunofluorescent microscopy with a Nikon Eclipse Ti-E microscope using Nikon NIS Elements software (Nikon Corporation). For quantification of TUNEL positivity, TUNEL foci were enumerated and then summed per square micron (200 magnification field). Enzyme activity assays Ceramide synthase. Ceramide synthase (CerS) activity was measured in 50 g of.Early acute kidney injury and sepsis: a multicentre evaluation. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. as adopted and promulgated by the US National Institutes of Health and were approved by the Institutional Animal Care and Use Committee. At 24 h post-glycerol injection, at the peak of injury (43), the kidneys were used for preparation of homogenate for measurement of sphingolipids and enzyme activities as described below. Measurement of kidney function BUN (40146) and SCr (C7548-120) levels were determined on serum samples using kits from AMS Diagnostics (Weston, FL) and Point Scientific Inc. (Canton, MI), respectively, following the manufactures instructions. Protein quantification and Western blot analysis Kidney cortex was homogenized in cell extraction buffer (Thermo Fisher Scientific, Waltham, MA), comprising a Complete Protease Inhibitor Cocktail tablet and a Phosphatase Inhibitor Cocktail tablet (Roche, Indianapolis, IN). Homogenates were centrifuged at 15,000 for 10 min at 4C. Supernatants were removed, combined, aliquoted, and stored at ?80C. Protein concentrations were identified using Bradford reagent (Bio-Rad, Hercules, CA). Kidney homogenate (40 g) was separated on 4C12% gradient Tris-glycine-SDS polyacrylamide gels and transferred to PVDF membranes and then clogged in 5% (w/v) dried milk in TBS with 0.1% Tween 20 (TBST) for 1 h. Membranes were incubated with main antibody over night at 4C. Membranes were then washed three times for 5 min each with TBST comprising 5% (w/v) dried milk. After incubation for 1 h at space temperature with secondary antibodies conjugated with HRP (1:10,000) in TBST comprising 1.25% (w/v) dried milk, membrane proteins were detected by chemiluminescence substrate. Antibodies were purchased from Cell Signaling (Beverly, MA) unless otherwise mentioned: inositol requiring enzyme-1 (IRE1) (#3294), ERK (#4695), phosphorylated (p)- ERK (#4370), cleaved caspase 3 (#9664), cleaved caspase 8 (#8592), C/EBP homologous protein (CHOP) (#2895), c-Jun N-terminal kinase (JNK) (#9258), phosphorylated-c-Jun N-terminal kinase (p-JNK) (#4668), proliferating cell nuclear antigen (PCNA) (#13110), and -actin (catalog quantity A2228; Sigma Aldrich). Gene manifestation RNA was isolated using TRIzol (Thermo Fisher Scientific) per the manufacturers protocol. cDNA was synthesized with high-capacity cDNA reverse transcriptase PCR (Thermo Fisher Scientific) per the manufacturers instructions. TNF- (Mm00443258_m1), interleukin (IL)-6 (Mm00446190_m1), chemokine (C-X-C motif) ligand 1 (CXCL1) (Mm04207460_m1), kidney injury molecule-1 (KIM-1) (Mm00506686), and the housekeeping gene, (LTA) like a proximal tubule marker. Briefly, kidney sections (5 m) were deparaffinized and rehydrated, antigens were unmasked, followed by obstructing of endogenous peroxidases. Sections were further clogged with 5% goat serum with 0.1% Triton X-100 in PBS, followed by incubation with HRP-conjugated LTA (1:500) for 2 h at space temperature. HRP-LTA was recognized with Nova Red peroxidase (SK-4800; Vector Labs, Burlingame, CA). Next, sections were clogged for avidin and biotin, followed by anti-PCNA (1:16,000) incubation immediately at 4C. Sections were then incubated with biotinylated goat anti-rabbit IgG antibody (1:25,000, BA-1000; Vector Laboratories) for 30 min at space temp. Vector ABC reagent (PK-7100; Vector Laboratories) was added to each section and incubated for 30 min at space temp. PCNA was then visualized with Nova Red DAB substrate (SK-4800; Vector Laboratories). Slides were counterstained with revised Mayers hematoxylin (72804; Thermo Fisher Scientific), and then dehydrated followed by mounting with Permount (SP15; Thermo Fisher Scientific). Images were visualized using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan). For quantification of PCNA, positive cells that stained brownish in color were enumerated and then summed per square micron (200 magnification field). TUNEL assays were performed on paraffin inlayed tissue sections using Apoptag Red in situ apoptosis detection kit per the manufacturers instructions (S7165;.