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. 2020 Sep 10;35(1):1697–1711. doi: 10.1080/14756366.2020.1818738

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© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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graphic file with name IENZ_A_1818738_F0001a_C.jpg — Effect of erythropoietin beta (Epo), LFM-A13 (LFM), and their combined activity on cell viability of MCF-7 (A) MDA-MB-231 (B); *p < .05 (vs. Con); ^p < .05 (vs. Epo); #p < .05 (vs. LFM-A13. Combination index analysis of erythropoietin (10–100 IU/ml) combined with LFM-A13 (10–100 µM) at a constant ratio in MCF-7 (C), and MDA-MB-231 (D) cells. Synergistic effects are defined as CI < 1, additive effects are CI = 1, and antagonistic effects are CI > 1. Schematic of xenograft assay and analysis of tumour development. Site-specific injection (yolk sac) of CM-Dil (red) breast cancer cells (MCF-7, MDA- MDA-MB-231) into 48 hpf zebrafish embryos and imaging analysis of tumour growth after 48 h incubation with combination of exogenous erythropoietin beta (100 IU/ml) and/or LFM-A13 (100 µM) (E, F). Quantification of total CM-DiI fluorescence by breast cancer cells 3 d after injection; n = 4, *p < .05 (G). Immunoblotting analysis for phospho-BTK (pBTK) and total BTK (BTK) in MCF-7 and MDA-MB-231 cells treated with erythropoietin beta (Epo 100 IU/ml), LFM-A13 (LFM 100 µM), and both for 48 h. Samples used for electrophoresis consisted of 20 µg of protein from 6 pooled cell extracts (n = 6). Band staining was quantified by densitometry. Bands of phospho-proteins were normalised to respective total proteins. BTK plus pBTK expressions were assessed in a pooled sample of three independent experiments (H).

graphic file with name IENZ_A_1818738_F0001b_C.jpg — Effect of erythropoietin beta (Epo), LFM-A13 (LFM), and their combined activity on cell viability of MCF-7 (A) MDA-MB-231 (B); *p < .05 (vs. Con); ^p < .05 (vs. Epo); #p < .05 (vs. LFM-A13. Combination index analysis of erythropoietin (10–100 IU/ml) combined with LFM-A13 (10–100 µM) at a constant ratio in MCF-7 (C), and MDA-MB-231 (D) cells. Synergistic effects are defined as CI < 1, additive effects are CI = 1, and antagonistic effects are CI > 1. Schematic of xenograft assay and analysis of tumour development. Site-specific injection (yolk sac) of CM-Dil (red) breast cancer cells (MCF-7, MDA- MDA-MB-231) into 48 hpf zebrafish embryos and imaging analysis of tumour growth after 48 h incubation with combination of exogenous erythropoietin beta (100 IU/ml) and/or LFM-A13 (100 µM) (E, F). Quantification of total CM-DiI fluorescence by breast cancer cells 3 d after injection; n = 4, *p < .05 (G). Immunoblotting analysis for phospho-BTK (pBTK) and total BTK (BTK) in MCF-7 and MDA-MB-231 cells treated with erythropoietin beta (Epo 100 IU/ml), LFM-A13 (LFM 100 µM), and both for 48 h. Samples used for electrophoresis consisted of 20 µg of protein from 6 pooled cell extracts (n = 6). Band staining was quantified by densitometry. Bands of phospho-proteins were normalised to respective total proteins. BTK plus pBTK expressions were assessed in a pooled sample of three independent experiments (H).