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. 2018 Jun 4;9:2165. doi: 10.1038/s41467-018-04607-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — ROCK blockade enhances processing of Dox-treated cells. a, b CellTracker Deep Red-labelled BMDCs were co-cultured with CMFDA-stained, Dox-treated CT26.CL25 or B16F10-Ova cells for 5 min, and then analysed by flow cytometry. a Representative flow cytometry plots. Upper right quadrant indicates BMDCs phagocytosing cancer cells. b The percentage of phagocytosis. Data are presented as means ± s.d. (n = 3). c, d CMFDA-labelled BMDCs were co-cultured with pHrodo-labelled CT26.CL25 cells for 5 min in the presence of Y27632 or vehicle, and analysed under a fluorescent microscope. c Representative images of BMDCs phagocytosing pHrodo-labelled, Dox-treated CT26.CL25 cells. CMFDA-labelled BMDCs are shown in green, and engulfed CT26.CL25 cells are shown in red. Scale bar, 50 μm. d Phagocytosis, calculated as the percentage of the total number of BMDCs containing cancer cells. Data are presented as means ± s.d. (n = 8). e, f BMDCs were co-cultured with Dox-treated CT26.CL25 cells for 4 h, and un-engulfed cells were then removed. After further incubation for 20 h, the expressions of CD40 and CD86 in BMDCs were analysed by flow cytometry. e Representative flow cytometry plots. The gray peaks represent the isotype control. f The percentage of CD40⁺ and CD86⁺ BMDCs. Data are presented as means ± s.d. (n = 3–4). g BMDCs were pretreated with Y27632 or vehicle and co-cultured with B16F10-Ova cells or Dox-treated B16F10-Ova cells for 4 h and then un-engulfed cells were removed. After further incubation for 20 h, the percentage of H-2k^b-OVA⁺ BMDCs was analysed by flow cytometry using an antibody recognizing a complex of the OVA peptide (SIINFEKL) with H-2k^b (MHC-I). Data are presented as means ± s.d. (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001; significance determined by unpaired Student’s t test (b, d, f), or one-way ANOVA with Tukey’s post hoc test (g)