Fig 1.
HF synergistically interacts with RAPA in the suppression of TCR stimulated T cell proliferation.
T cell proliferation in response to the stimulation of anti-CD3 antibody binding to TCR complex was measured by using MTT assay or trypan blue exclusion. (A) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 1 nM of RAPA for 48 hrs. (B) Anti-CD3 antibody-stimulated splenocytes were treated with RAPA alone or in combination with 2.5 nM of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments, and were statistically analyzed using two-way ANOVA. P < 0.0001 (HF vs. HF + RAPA); P < 0.0001 (RAPA vs. RAPA + HF). γ = 0.1905 based on IC70 values in these two experiments. (C) Anti-CD3 antibody-stimulated splenocytes were treated with HF (1 or 2 nM) or RAPA (0.5 nM) alone or in combination with both drugs for 48 hrs, followed by counting viable cells using trypan blue exclusion assay. Data are presented as mean ± SD of three experiments. The Bliss independence: τ < α. (D) The splenocyte cultures were treated with HF alone, RAPA alone or both HF and RAPA for 18 hrs, and the cell growth was determined by using MTT assay. The difference of inhibition between groups was analyzed using two-tailed t-test. Data are presented as mean ± SD of five experiments. The Bliss independence: τ < α. (E) The splenocyte cultures were treated with HF alone or in combination with 1 nM RAPA for 36 hrs. (F) The splenocyte cultures were treated with RAPA alone or in combination with 2.5 nM HF for 36 hrs. The cell growth was determined using MTT assay. Data are presented as mean ± SD of five experiments. The difference of drug inhibitions between groups was analyzed using two-way ANOVA. P < 0.0001 (HF vs. HF + RAPA); P < 0.0001 (RAPA vs. RAPA + HF). γ = 0.201 based on IC70 values in these two experiments.
Table 1.
The interaction of RAPA with HF in the suppression of cell growth in T cell cultures.
Fig 2.
Excess proline does not interfere synergistic interaction of RAPA with HF in the suppression of T cell proliferation.
Anti-CD3 antibody-stimulated naïve splenocytes were treated with HF (2.5 nM) alone, RAPA (1 nM) alone or a mixture of RAPA (1 nM) and HF (2.5 nM) in the presence of 1 mM proline or vehicle for 48 hrs. T cell proliferation was measured by using MTT assay. Data are presented as the means ± SD of three separate experiments, and were statistically compared between vehicle and proline-treated samples in each group by two-tailed t-test.
Fig 3.
RAPA potentiates HF-induced cell death in FACS analysis.
Cell death in cultured splenocytes was determined by FACS analysis with annexin-V-PE and 7-AAD staining. (A) Anti-CD3 antibody-stimulated splenocytes were treated with 1 nM of RAPA alone or in combination with various concentrations of HF for 48 hrs. Data are presented as a typical dot plot showing the percentage of annexin-V-PE and/or 7-AAD positive staining of cell populations. (B) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 1 nM of RAPA for 48 hrs. (C) Anti-CD3 antibody-stimulated splenocytes were treated with RAPA alone or in combination with 2.5 nM of HF for 48 hrs. Apoptosis was represented by the sum of annexin-V stained cell populations (single annexin-V-PE positive cells in lower right quadrant, and double-annexin-V-PE/7-AAD positive cells in upper right quadrant). Data are presented as mean ± SD of 3–4 separate experiments, and were statistically analyzed by ANOVA. P ˂ 0.0001 (HF vs. HF + RAPA, n = 3); P ˂ 0.0001 (RAPA vs. RAPA + HF, n = 4).
Fig 4.
RAPA potentiates HF-induced cell death in Western blot.
The cleaved form of PARP as a biomarker for cell apoptosis in protein extracts of splenocytes was analyzed by Western blot. Anti-CD3 antibody-stimulated splenocytes were treated with RAPA (1 nM) alone, HF (2.5 nM) alone or a mixture of RAPA (1 nM) and HF (2.5 nM) for 48 hrs. Equal amount of protein (approximately 250 μg) extracted from whole cell pellets was fractioned by 7% of SDS-PAGE, and the cleaved PARP protein bands was identified based on specifically binding of anti-cleaved PARP antibody, and their molecular size (cleaved PARP: 89 kDa). The protein content in each sample was confirmed by re-probing the blot with anti-β-actin IgG antibody and was measured by a densitometry. Imaging data are a representative of three separate experiments. The ratio unit (RU) of PARP band to actin band from the same sample on the same blot was presented as mean ± SD of three determinants. P ˂ 0.0001 (HF vs. HF + RAPA), P ˂ 0.0001 (RAPA vs. HF + RAPA), P = 0.0009 (HF vs. RAPA).
Fig 5.
HF antagonistically interacts with CsA in the suppression of TCR stimulated T cell proliferation.
T cell proliferation in response to the stimulation of anti-CD3 antibody binding to TCR complex was measured by MTT assay. (A) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 5 nM of CsA for 48 hrs. (B) Anti-CD3 antibody-stimulated splenocytes were treated with CsA alone or in combination with 2.5 nM of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments, and were statistically analyzed by ANOVA. P = 0.0014 (HF vs. HF + CsA); P ˂ 0.0001 (CsA vs. CsA + HF). γ = 1.8375 based on IC50 values in these two experiments.
Fig 6.
HF reduces CsA-induced cell death in cultured HK-2 cells.
Cell viability or apoptosis was measured by FACS analysis with annexin-V-PE and 7-AAD staining. A monolayer of HK-2 cells was treated with CsA (0–10 μM) alone or in combination with 1 μM HF for 48 hrs. Cell viability represented the percentage of viable cells (double-annexin-V-PE/7-AAD negative cells in lower left quadrant), and apoptosis was the sum of Annexin-V stained cell populations (single annexin-V-PE positive cells in lower right quadrant, and double-annexin-V-PE/7-AAD positive cells in upper right quadrant). Data are presented as mean ± SD of three separate experiments, and were statistically compared between CsA alone and CsA with HF in each group by two-tailed t-test.