FIGURES AND TABLES

Figure 1.

Effect of cyclosporine (CsA) on DNA and protein synthesis in LLC-PK₁ cells. (A)³H-thymidine incorporation was used as an index of DNA synthesis. (B)¹⁴C-leucine incorporation was used as an index of protein synthesis. In both cases results are expressed as the DPM of radioisotope incorporated per well and represent the mean SEM of 3 independent experiments each performed in quadruplicate. *P < 0.05, **P < 0.01.

Figure 2.

Effect of cyclosporine (CsA) on trypan blue exclusion and LDH release in LLC-PK₁ cells. LLC-PK₁ cells were exposed to 4.2 nM and 42 M CsA for 24 hours. (A) For trypan blue exclusion (% membrane integrity) cells were trysinized, mixed with trypan blue and viewed under a light microscope. Cells that were blue were scored as having an altered membrane integrity. (B) Fractional release of LDH was determined by the equation S/(S + C); where S = LDH in the supernatant and C = LDH in the monolayer, after cell lysis. Results represent the mean SEM of 3 to 6 independent experiments. **P < 0.01.

Figure 3.

Flow cytometric analysis of the effect of cyclosporine (CsA) treatment for 24 hours on the DNA profile, size and granularity of LLC-PK₁ cells. Results from one representative experiment that represents 10,000 events (cells) are shown. (A) DNA profile of control cells. (B) DNA profile of 4.2 nM CsA treated cells. (C) DNA profile of 4.2 M CsA treated cells. (D) Light scattering properties of control cells; FSC, size and SSC, granularity. (E) Light scattering properties of 4.2 nM CsA treated cells. (F) Light scattering properties of 4.2 M CsA.

Figure 4.

Apoptosis detection following cyclosporine (CsA) treatment using the TUNEL assay. Cells were treated for 24 hours with 4.2 nM CsA and apoptotic cells were detected using the TUNEL assay. Arrows indicate TUNEL positive cells. (A) Cells treated with vehicle alone. (B) Cells treated for 24 hours with 4.2 nM CsA.

Figure 5.

Flow cytometric assessment of the effects of a low and high dose of cyclosporine (CsA) on the size and granularity of LLC-PK₁ cells. Forward light scatter (FSC) and side light scatter (SSC) of the laser beam were used as indices of size and granularity, respectively. Results from one representative experiment which represent 10,000 events (cells) are shown. (A) Control. (B) 4.2 nM CsA. (C) 42 M CsA.

Figure 6.

FITC-annexin V binding in LLC-PK₁ cells following cyclosporine (CsA) exposure. Cells were treated for 24 hr with 4.2 nM CsA. The externalization of phosphatidylserine was assessed by measuring FITC-annexin V binding. The far right peak represents cells which have bound FITC-annexin V. One representative experiment out of 3 is shown.

Figure 7.

Effect of cyclosporine (4.2 nM CsA) on the expression of Fas in LLC-PK₁ cells. Cells were treated for 24 hours with 4.2 nM CsA, and Fas expression was assessed using flow cytometric immunofluoresence and Western blotting technqiues as described in the Methods section. (A) Graphical representation of Fas expression as assessed by flow cytometric immunofluoresence. Results are expressed as the percentage of control cell Fas expression and represent the mean SEM of 3 independent experiments. *P < 0.05. (B) Flow cytometric histogram illustrating the increase in Fas expression following CsA treatment. (C) Western blot analysis of Fas expression.

Figure 8.

Effect of the Fas antibody in the presence and absence of cyclosporine (CsA) on the light scattering properties of LLC-PK₁ cells. (A) Light scattering properties of control cells incubated in the absence of primary antibody (-/+). (B) Light scattering properties of control cells incubated with both primary and secondary antibody (+/+). (C) Light scattering properties of CsA treated cells incubated with both primary and secondary antibody.

Table 1 - Dose-response effects of cyclosporine (CsA) on the cell cycle and apoptosis in LLC-PK₁ cells.