1. Volume 117, Issue 4, Pages 848-857 (October 1999)
Distinct protein kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells 
Shaun G. Weller, Irene K. Klein, Robert C. Penington, William E. Karnes 
Gastroenterology 
Volume 117, Issue 4, Pages (October 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A) Concentration-dependent effects of PMA on viable cell mass of colon cancer cell lines detected by MTT assay. The y-axis shows the fraction of the mean absorbance values (Abs 492) after 72 hours for each treatment compared with plated controls at time 0; x-axis shows concentrations of PMA (nmol/L). Data are from a representative experiment repeated 3 times. Each value is calculated from 8 identically treated wells (including controls). Standard deviations were consistently <5% of the mean (not shown). Colon cancer cell lines are SNU-C1 (●), SNU-C4 (■), SNU-C5 (♢), SW480 (2), and HCT116 (○). (B) Concentration-dependent effects of PMA, IDB, and bistratene A on viable cell mass of SNU-C4 cells detected by MTT assay. The y-axis shows fraction of the mean absorbance values (Abs 492) after 72 hours for each treatment compared with plated controls at time 0; x-axis shows concentrations of agonists (nmol/L). Data are from a representative experiment repeated 3 times. Each value is calculated from 8 identically treated wells (including controls). Standard deviations were consistently <5% of the mean (not shown). Agonists are PMA (●), IDB (▴), and bistratene A (♦).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
(A) Concentration-dependent effects of PMA on viable cell mass and DNA fragmentation of SNU-C1 and SNU-C4 cells. Left y-axis shows fractional change of mean MTT absorbance values compared with that of untreated cells after 48-hour treatment with PMA from 8 identically treated wells (SNU-C1, ♦; SNU-C4, ■). Right y-axis shows mean absorbance values of the DNA-fragmentation ELISA (Abs 450), reflecting the concentration of enzymatically labeled 3' DNA ends after 24-hour treatment with PMA from 8 identically treated wells (SNU-C1, ♢; SNU-C4, 2). The x-axis shows concentrations of PMA (nmol/L). Data are from a representative experiment repeated 3 times. Standard deviations were consistently <5% of the mean for the MTT assay and <10% for the DNA-fragmentation ELISA (not shown). (B) Effects of PMA on morphology and nuclear fragmentation on SNU-C4 cells. Representative fields from wells treated with diluent (control), 1 nmol/L PMA, 10 nmol/L PMA, and 10 μmol/L bistratene A after 48 hours (original magnification 200×). Upper row shows transmission light microscopy. Lower row shows corresponding fluorescent nuclear staining using Hoescht Representative apoptotic cells with fragmented nuclei are shown by arrows.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
(A) Time-dependent effects of PMA (10 nmol/L) on percent apoptosis of SNU-C4 cells as detected by flow cytometry (annexin V binding) and by nuclear staining with Hoescht The y-axis shows percent apoptosis as detected by nuclear fragmentation using Hoescht staining (○; minimum of 300 total cells counted) or annexin V–bound cells that excluded propidium iodide (●; 20,000 cells counted). The x-axis shows treatment time (hours). Data for annexin V and Hoescht are from representative experiments repeated 3 times. (B) Concentration-dependent effects of GF X on PMA-induced apoptosis in SNU-C4 cells. The y-axis shows percent apoptosis was detected by annexin V binding after 12-hour treatment with 10 nmol/L PMA as described for figure A; x-axis shows GF X concentration (nmol/L). Data are from representative experiments repeated 3 times. Inset shows representative fluorescent photomicrographs of Hoescht 33342–stained SNU-C4 cells treated with 10 nmol/L PMA with and without 1 μmol/L GF X for 12 hours.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
(A) Effects of PMA and GF X on mitogenesis detected by [3H]thymidine incorporation. The y-axis shows mean degradations per minute (dpm) of [3H]thymidine in precipitated DNA from 4 identically treated samples. Error bars represent standard deviation of the mean. The x-axis shows treatments with or without PMA (1 nmol/L), varying concentrations of GF X (nmol/L), and effects of EGF in the presence of 1 nmol/L PMA and 3 μmol/L GF X. Data are from a representative experiment repeated 3 times. (B) Effects of PMA and GF X on morphology and nuclear fragmentation of SNU-C4 cells. Representative fields from wells treated with diluent (CNTL), 1 nmol/L PMA alone (PMA), or 1 nmol/L PMA plus 3 μmol/L GF X (PMA+GF) after 48 hours (original magnification 200×). Left column shows transmission light microscopy; right column shows corresponding fluorescent nuclear staining using Hoescht
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Concentration-dependent effects of (A) PMA, (B) IDB, and (C) bistratene A on cytosolic and membrane levels of PKC isozymes in SNU-C4 cells as detected by immunoblotting. Cells were treated for 15 minutes and processed as described in Materials and Methods. PKC isozymes to which each antibody was raised are labeled on the left. Agonist concentrations are shown at the bottom of each panel: PMA (nmol/L), IDB (μmol/L), and bistratene A (μmol/L). Data are representative of at least 3 separate experiments showing identical effects.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Concentration-dependent effects of PMA on levels of PKC isozymes in SNU-C4 cells as detected by immunoblotting. Total cell lysates were prepared as described in Materials and Methods. Treatments with diluent (CTRL) and 1 or 10 nmol/L PMA for 24 hours are shown on the top. Isozymes detected by immunoblotting are labeled on the left. Data are representative of 3 separate experiments showing identical effects.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions