Figures & data
Notes: Human U937 (A, B), Jurkat (C), and KU812 (D) cells were incubated with either DMSO (vehicle control) or 0.5–10 µM lapatinib for 1–3 days as indicated. The relative percentages of survival rates were analyzed by MTS (A, C, D) or trypan blue (B) assays. Mean OD values or cell numbers of vehicle control were defined as 100%. *P<0.05, **P<0.01, and ***P<0.001 (Student’s t-test) as compared to the DMSO control.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; OD, optical density.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; OD, optical density.
Notes: Human U937 (A–C), MEG-01 (D), NB4 (E), and Jurkat (F) cells were either untreated (unTx) or treated with DMSO (D, vehicle control), 10 µM (A), or 2.5–10 µM lapatinib as indicated for 3 days or 2–3 days (C). (A) The percentage of dead cells and sub-G1 cells were measured side-by-side by flow cytometry. (C–F) After the treatment of cells as indicated, the percentage of sub-G1 (hypodiploid or apoptotic cells) was measured by flow cytometry. (B, C, F) Cells were treated with lapatinib in the presence or absence of 20 µM pan-caspase inhibitor Z-VAD-fmk; cell viability (B) or percentage of sub-G1 cells (C, F) was analyzed by MTS assay (B) or flow cytometry (C, F). *P<0.05 and ***P<0.001 (Student’s t-test) as compared to the DMSO control (A, D, E) or lapatinib plus Z-VAD-fmk treatment (B, F). “–” in the absence of Z-VAD-fmk.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; unTx, untreated.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; unTx, untreated.
Notes: (A) The induction of vacuole LC3 aggregations by lapatinib. U937 cells were treated with DMSO, 5–10 µM lapatinib, or 0.1 µM TPA (differentiation inducer) as indicated for 3 days. Cells were stained with Liu’s stain. The morphology of cells was observed. (B) The induction of vacuole LC3 aggregations by lapatinib. GFP-LC3-overexpressing U937 cells were treated with DMSO, 5–10 µM lapatinib, or 150 nM thapsigargin (autophagy inducer) as indicated for 3 days. Cells with LC3 aggregations were observed by fluorescence microscopy. (C) U937 cells were treated with DMSO or 2.5–5 µM lapatinib for 3 days. Cells were stained with acridine orange and analyzed by flow cytometry. (D) Cells were treated with lapatinib in the presence or absence of 1.25 mM autophagy inhibitor 3-MA; cell viability was analyzed by an MTS assay. ***P<0.001 (Student’s t-test) as compared to lapatinib plus 3-MA treatment. (E) U937 cells were treated with DMSO or 2.5–10 µM lapatinib for 24 hours (left panel) or 4–24 hours as indicated. Cell lysates were collected and subjected to Western blot analysis for ATG7, ATG5, Beclin-1, BNIP, LC3, and actin (loading control). “–” in the absence of 3-MA.
Abbreviations: DMSO, dimethyl sulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate; TG, thapsigargin; 3-MA, 3-methyladenine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Abbreviations: DMSO, dimethyl sulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate; TG, thapsigargin; 3-MA, 3-methyladenine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Notes: U937 cells were treated with (A) DMSO or (B) 10 µM lapatinib for 3 days. Cells were fixed, stained, and observed by TEM. The magnification information of each figure is as indicated.
Abbreviations: DMSO, dimethyl sulfoxide; TEM, transmission electron microscope.
Abbreviations: DMSO, dimethyl sulfoxide; TEM, transmission electron microscope.
Notes: U937 cells with shRNA expression were prepared. Cells were transduced with shRNA against ATG5, 7, Beclin-1 (BECN), and RFP (nonspecific) mRNA, selected with puromycin, and treated with DMSO(D) or 5–10 µM lapatinib for 2 days. The relative percentages of survival rates were detected using the MTS (A) and trypan blue (B) assays. Two shATG7 or shBECN clones (#1 or #2) were used. *P<0.05, **P<0.01, and ***P<0.001 (Student’s t-test) as compared to the DMSO control.
Abbreviations: RNAi, RNA interference; shRNA, short hairpin RNA; DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Unif, uninfected cells.
Abbreviations: RNAi, RNA interference; shRNA, short hairpin RNA; DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Unif, uninfected cells.
Notes: (A, B) U937 cells were treated with DMSO, 5–10 µM lapatinib, or 0.1 µM TPA (differentiation inducer) for 3 days. Cells were harvested, stained with antibodies against CD14 or CD68, and positive cells were analyzed by flow cytometry. (C–E) U937 cells were treated with DMSO, 2.5–10 µM lapatinib, or 0.1 µM TPA (differentiation inducer) as indicated for 3 days. Cells were harvested, incubated with fluorescent latex beads (C, D) or DCFH-DA (E) for the detection of the phagocytosis ability of cells (C, D) or the ROS production of cells (E) by flow cytometry. (D) MFI in (C) is expressed. ***P<0.001 (t-test) as compared to the DMSO control.
Abbreviations: AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ROS, reactive oxygen species; MFI, mean fluorescence intensity.
Abbreviations: AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ROS, reactive oxygen species; MFI, mean fluorescence intensity.
Notes: U937 cells were treated with DMSO, 1.25–5 µM lapatinib, 20–40 µM erlotinib, 0.5–5 nM etoposide, 1–25 nM paclitaxel, or a combination of two drugs as indicated for 3 days. Cell viability was analyzed by MTS. **P<0.05 and ***P<0.001 (Student’s t-test) as compared to 1.25 or 5 µM lapatinib alone treatment.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.