Fig 2: Cortactin and N-WASP are recruited on Circular Dorsal Ruffles (CDRs) that are transient PM structures induced by Trastuzumab (Tz)(A) SK-BR-3 were treated with Tz for 15 minutes, fixed and processed for immunofluorescence. (a) F-Actin stained with fluorescent phalloidin; (b) Cortactin revealed with a specific antibody; (c) N-WASP:GFP chimera revealed in the green channel; (d, e, f) ERBB2 detected with anti-ERBB2 antibody. Arrowheads indicate colocalization of F-actin, cortactin and N-WASP:GFP green signals with ERBB2 red signals in CDRs (g, h, i). Scale bars = 10 μm. (B) Quantification of Tz-induced CDR formation in SK-BR-3 control cells (NT) and SK-BR-3 cells treated with Tz at various time points indicated in min. Bars represent the average ± SD of the data pooled from 3 independent experiments. At each time point at least 500 cells were analyzed.

Fig 3: Representative fluorescent images of SKBR3 cells maintained in tunicamycin (1 μg/ml) and stained with WGA conjugated QD at 4 °C for 3 h and SKBR-3 cells maintained in medium without tunicamycin and treated with the same volume of QD alone (A). The percentage survival of SKBR-3 cells maintained with/without tunicamycin and following treatment with DXR (1 nM–10 μM) the mean % cell survival was calculated assuming 100% survival for untreated cells (B). Cell stimulation as measured by % cell count of SKBR-3 cells maintained with/without tunicamycin and exposed to EGF, IGF alone or in combination (C). Western blot analysis of EGFR (HER1) and HER2 for SKBR-3 cells pre and post treatment with tunicamycin a faint band which may represent deglycoyslated HER2 in the tunicamycin treated cells is shown by the arrow (D). UT: Untreated, Tx: tunicamycin treated GAPDH levels were assessed as a gel loading control.

Fig 4: Melatonin induces ubiquitylation of endocytosed HER2 through reducing the expression of HSP90 and its association with HER2.A Western blot analysis of proteins as indicated in the HCC1954 cells treated with or without Melatonin. Drug treatment conditions were shown as indicated. Vinculin was used as a loading control. The quantification of protein abundance is shown. B Co-Immunoprecipitation (Co-IP) of HER2 and HSP90 from the lysates of the Melatonin-treated HCC1954 cells or control cells. Melatonin, 2 mM, 48 h. Co-immunoprecipitated HSP90 and ubiquitylated HER2 band intensities were quantified and adjusted to the corresponding normalized HER2 signal and shown in the bar charts. Data from three independent experiments are shown as Mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). C Representative images of immunofluorescent staining of HER2 and EEA1 proteins in the HCC1954 cells treated with 2 mM Melatonin for 24 h. D Representative images of immunofluorescent staining of HER2 and LAMP1 proteins in the HCC1954 cells treated as in C. Scale bar, 25 μm.

Fig 5: Melatonin synergizes with HER2 inhibitor Neratinib to induce apoptosis and cytotoxicity in vitro.Long-term cell viability of HER2+ breast cancer cells treated with Melatonin and Neratinib, either alone or in combination, was examined by crystal violet assay. HCC1954, 2 mM Melatonin, 50 nM Neratinib; MDA-MB-453, 2 mM Melatonin, 100 nM Neratinib; MDA-MB-361, 2 mM Melatonin, 100 nM Neratinib; MCF7/HER2, 1 mM Melatonin, 50 nM Neratinib. A, 96-well format; B, 24-well format. Representative images of plates and the quantification of cell viability are shown. C Cell migration potential of the HER2+ breast cancers cells treated as in A was determined using a wound-healing assay. The images of the wound areas are shown at 0 and 48 h. Scale bar, 200 μm. D Western blot analysis of proteins as indicated in cells treated as in A for 24 h. Vinculin was used as a loading control. The quantification of protein abundance is shown. E Cell death in the HER2+ breast cancer cells treated as in A for 48 h was examined by PI staining and FACS analysis. Data from three independent experiments are shown as Mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).