Fig. 4. Tocilizumab prevents Cisplatin-induced self-renewal of cells in orospheres.

A Representative images of immunofluorescence staining of untreated, cryosectioned orospheres after 8 days in suspension culture. Example images of both a large (left) and small (right) orosphere are shown. B Bar graphs depicting the mean number ± SD of primary orospheres per well (n = 6 per treatment group). Different lowercase letters indicate statistical significance at p < 0.05. C Representative images (×40) of primary orospheres 8 days after treatment with Cisplatin (1 µm) and/or Tocilizumab (0.1 µm). Cells were treated the day after seeding in ultra-low attachment plates. Inserts at ×100 magnification. D Bar graphs depicting the mean number ± SD of secondary orospheres per well (n = 6 per treatment group). Different lowercase letters indicate statistical significance at p < 0.05. E Representative images (×40) of secondary orospheres on day 8 after treatment with Cisplatin (1 µM) and/or Tocilizumab (0.1 µM). Cells were treated the day after seeding in ultra-low attachment plates. Inserts at ×100 magnification. F Western blottings showing protein levels Bmi-1, IL-6R, gp130, pSTAT3, and STAT3 in lysates prepared from primary orospheres on day 8 after treatment with Cisplatin (1 µM) and/or Tocilizumab (0.1 µM).