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Figure 1 | Molecular Cancer

Figure 1

From: Differential expression and role of p21cip/waf1 and p27kip1 in TNF-α-induced inhibition of proliferation in human glioma cells

Figure 1

A-F. TNF-α-induced inhibition of proliferation in LN-18 cells is mediated by TNFR1. (A) LN-18 cells cultured as monolayers and spheroids for 24 hr were treated with serial concentrations of TNF-α for 24 hr, during the last 18 hr of treatment, 1 μCi of triatiated thymidine was added to the wells. The cells were harvested and the radio activity-counts per minute (CPM) was measured and percent inhibition of proliferation was calculated considering the CPM in untreated cells as 100%. B) Expression of TNFR1 and TNFR2 was analyzed by RT-PCR in LN-18 cells and compared with human glioma cell line-U87MG, used as positive control. β-actin was used as control. C) Expression of TNFR1 and TNFR2 in LN-18 cells and U87MG cells analyzed by flowcytometry, bold lines represent TNFR1/TNFR2 expression and dotted lines depict profiles with isocontrol antibody. D) Monolayers and spheroids were preincubated with neutralizing antibodies to TNFR1 and TNFR2 (50 μg/ml) or isocontrol antibody for 2 hr and treated with TNF-α (10 ng/ml) for 24 hr and proliferation assay was done as described above. E) Monolayers and spheroids were treated with serial concentrations of TNFR1 agonistic antibody or isocontrol antibody for 24 hr and proliferation was determined by triatiated thymidine incorporation assay. The data for A, D and E is the mean ± SE of at least three independent experiments done in triplicates. * p < 0.05-comparison of inhibition of proliferation in TNF-α treated cells in the presence and absence of TNFR neutralizing antibodies for D and between treated and controls cells for A and E. F) Levels of TNFR1 expression by Western blotting in monolayers (M) and spheroids (S) cultured for 24 hr. The blots were stripped and reprobed with β-actin, which served as a loading control of proteins.

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