SUPPLEMENTAL INFORMATION
Supplemental material and methods
Antibodies and Inhibitor :
Monoclonal p21 was from Santa Cruz Biotechnology (TEBU; Le Perray en Yvelines, France). Monoclonal cyclin D1 and pRb were from BD Bioscience (Erembodegem, Belgium). CXCR2 inhibitor SB 225002 was from Tocris (Bristol, UK).
Small interfering RNA-mediated downregulation of PPARg
Small interfering RNAs (siRNAs) were from Santa Cruz Biotechnology (sc-29455). As a nonspecific control, a scramble sequence for PPARg siRNA was used. siRNA duplex were transfected into cells using Lipofectamin RNAiMAX (Invitrogen). Transfections were carried out in six-well plates at 50–60% confluency with a final concentration of 20 nM of siRNA, and cells were harvested after 72 hours.
SUPPLEMENTAL RESULTS
Effect of PPARg depletion on ciglitazone-induced decrease of CXCL1 and MITF.
To assess the implication of PPARg receptor in the effects of ciglitazone on CXCL1 and MITF decrease, we silenced PPARg using siRNAs. Figure S1A shows that PPARg depletion does not prevent CXCL1 downregulation induced by ciglitazone treatment suggesting that ciglitazone effect on CXCL1 downregulation is independent of the activation of PPARg. Similarly, MITF downregulation in response to ciglitazone treatment monitored by western blot seems to be a PPARg independent event (Fig. S1B).
Effect of rCXCL1 on cell cycle arrest induced by ciglitazone
We have previously established that low doses of ciglitazone induce a G0/G1 cell cycle arrest of A375 melanoma cells. In order to determine if the decrease of CXCL1 is important in cell cycle arrest mediated by ciglitazone, we studied whether rCXCL1 protects the cells from the anti-proliferative effect of ciglitazone. For this, we analyzed the percentage of A375 cells in each phase of the cell cycle by flow cytometry analysis. Ciglitazone (0.1 and 1µM) decreased by 55% the proportion of cells in S phase and increased by 16% the number of cells in G0/G1 phase (Fig. S3A). After rCXCL1 treatment, low ciglitazone concentrations had a weak effect on cell cycle arrest. We observed a decrease by 15% in cells in S phase and an increase by 7% in G0/G1-phase cells. 10µM ciglitazone decreased the proportion of cells in S and G0/G1, and increased dramatically the SubG1 population (apoptotic cells). This cell population in SubG1 disappeared when A375 cells were treated with rCXCL1.
We next analyzed the expression and phosphorylation of proteins involved in the regulation of cell cycle (Fig. S3B). We observed a significant reduction of cyclin D1 when cells were treated by low concentrations of ciglitazone (0.1 and 1µM) associated with accumulation of the CDKI, p21 and a reduction in Rb phosphorylation. After addition of rCXCL1, the increase in p21 expression and the reduction of cyclin D1 were not detectable anymore and Rb was still hyperphosphorylated. As observed on western blot, induction of apoptosis by 10µM ciglitazone led to the degradation of these three proteins that were described as caspase 3 substrates (Katsuda et al., 2002; Woo et al., 2003). Consistent with the protective effect of rCXCL1 on ciglitazone-induced apoptosis, addition of rCXCL1 prevented the degradation of these proteins. Thus, considering all our results, it seems that recombinant CXCL1 prevents the cell cycle arrest induced by ciglitazone and confirms the pro-survival role of CXCL1 in melanoma cells.
Effect of CXCR2 signaling inhibition on viability of A375 cells
We examined the involvement of CXCR2 signaling to verify that CXCL1 promoted autocrine pathway through its receptor CXCR2. When cells were incubated with increasing concentrations of neutralizing CXCR2 antibody (anti-CXCR2 Ab), a dose-dependent reduction of A375 cell viability was observed (Fig. S4A). In the same manner, a dose-dependent reduction of A375 cell viability was observed in response to the specific CXCR2 pharmacological inhibitor, SB225002 (Fig. S4B).
SUPPLEMENTAL FIGURE LEGENDS
Figure S1: Decrease of CXCL1 and MITF expression mediated by ciglitazone are PPARg independent events.
SK-Mel-28 melanoma cells were transfected with siRNA targeting PPARg or a control siRNA. Cells were then starved and treated with different concentrations of ciglitazone for 24 hours.
a) ELISA of CXCL1 was performed on supernatants from starved SK-Mel-28 melanoma cells treated as indicated.
b) MITF expression and PPARg silencing were analyzed by western blotting. HSP60 was used as a loading control. One representative experiment of three is shown. Significantly different from the corresponding control * p< 0.05; *** p< 0.001.
Figure S2: Apoptosis induction in WM793 melanoma cells in response to ciglitazone treatment.
Starved WM793 melanoma cells were treated or not with 10µM ciglitazone (Cigli.) for 24 hours. Proteins were separated by 7.5% SDS-PAGE and analyzed by western blot using the indicated antibody. HSP60 was used as loading control. One representative experiment of three is shown.
Figure S3: Recombinant CXCL1 is sufficient to abrogate cell cycle arrest induced by ciglitazone.
Starved A375 melanoma cells were treated for 24 hours with various concentrations of ciglitazone (Cigli.) added or not with 50 ng/ml of recombinant CXCL1 (rCXCL1).
a) Cells were detached and their DNA content was measured by flow cytometry. The table displays the percentage of cells in G0/G1 (non cycling cells), S (DNA replicating cells), G2/M (dividing cells) and Sub G1 stage (apoptotic cells). One representative experiment of three is shown.
b) Proteins (30µg per line) were separated by 12% SDS-PAGE and analyzed by western blot using the indicated antibody. HSP60 was used as loading control. One representative experiment of three is shown.
Figure S4: Inhibition of CXCR2 signaling using blocking antibody targeting CXCR2 or using chemical inhibitor of CXCR2 (SB225002) decreases cell viability.
a) XTT activity was performed on starved A375 cells treated for 24 hours with various concentrations of blocking antibody targeting CXCR2. Results are expressed in percent of control (100%). Data are mean +/- SD of three independent experiments performed in triplicate. Significantly different from the corresponding control ** p< 0.01; *** p< 0.001.
b) Cell counting using trypan blue was performed on starved A375 cells treated for 72 hours with various concentrations of chemical inhibitor of CXCR2 (SB225002). Results are expressed in percent of control (100%). Data are mean +/- SD of three independent experiments performed in triplicate. Significantly different from the corresponding control * p< 0.05; *** p< 0.001.
Figure S5: Ciglitazone-induced apoptosis is reversed by ZVAD-FMK.
Starved SK-Mel-28 melanoma cells were treated or not with 10µM ciglitazone added or not with 100µM Z-VAD-FMK for 24 hrs. Cells were then harvested and counted using trypan blue. Results are expressed in percent of control (100%). Significantly different from the corresponding control ** p< 0.01.
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