lc3 5f10  (Developmental Studies Hybridoma Bank)

Journal: International Journal of Oncology

Article Title: Drug resistance-related sunitinib sequestration in autophagolysosomes of endothelial cells

doi: 10.3892/ijo.2019.4924

Figure Lengend Snippet: Induction of drug resistance in HMEC-1 cells and sunitinib-induced activation of autophagy. (A) Replication times of cultured HMEC-1 cells in the presence of indicated concentrations of sunitinib. Data were calculated from more than two repetitions. (B) Evolution of the IC50 of sunitinib in HMEC-1 cells after treatment with 6 µ M sunitinib for different time periods. (C) Plotted survival rates of HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells. Normal HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells were exposed to the indicated concentrations of sunitinib for 72 h, and the cells were trypsinized and counted. Non-treated cells were set as 100%. (A-C) Curves are derived from the means of more than three experiments. (D) LysoTracker DND-99 density of HMEC-1 cells following incubation with 6 µ M sunitinib for different time periods. Cells were treated with sunitinib for 24 or 48 h and LysoTracker DND-99 was added followed by flow cytometric analysis. (E) LysoTracker DND-99 density of sunitinib-treated HMEC-1 cells and HMEC 6 µ M cells. The cells were washed five times and then incubated with LysoTracker DND-99. Fluorescence was measured with the control cells without LysoTracker DND-99 staining using flow cytometry. (F-H) Western blot analysis using anti-LAMP-1, anti-LC3 and anti-p62 antibodies. HMEC-1 and HMEC 6 µ M cells were exposed to 6 µ M sunitinib, or autophagy inducer 5 µ M rapamycin, or Hanks' media for 24 h. Subsequently, the cells were collected and extracted proteins were used for western blotting. The experiments were repeated at least three times. * P<0.05 vs. control HMEC-1 cells. (I) Colocalization of LC3 protein labeling with green sunitinib fluorescence. HMEC-1 and HMEC 6 µ M cells were treated with 6 µ M sunitinib for 24 h. Subsequently, the cells were stained with anti-LC3 (red fluorescence) (magnification, ×100). DAPI was added to stain the nuclei. (J) Colocalization of LAMP-1 protein labeling with green sunitinib fluorescence. HMEC-1 and HMEC 6 µ M cells were treated with 6 µ M sunitinib for 24 h. Subsequently, the cells were stained with anti-LAMP-1 (red fluorescence) (magnification, ×100). DAPI was added to stain the nuclei. LAMP-1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1A/1B-light chain 3; SQSTM1, sequestosome 1; Suni, sunitinib.

Article Snippet: Anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. 0231-100/LC3-5F10) was obtained from Enzo Life Sciences, Inc.; anti-lysosomal-associated membrane protein 1 (LAMP-1; H4A3; cat. no. sc-20011) was purchased from Santa Cruz Biotechnology, Inc.; anti-sequestosome 1 (SQSTM1)/p62 (cat. no. 5114) was obtained from Cell Signaling Technology, Inc.; horseradish peroxidase-labeled anti-rabbit/mouse IgG antibodies (cat. no. 31460/31430) were obtained from Invitrogen; Thermo Fisher Scientific, Inc.; anti-β-actin (cat. no. A5316) was purchased from Sigma-Aldrich; Merck KGaA; goat anti-mouse IgG and goat anti-rabbit IgG coupled to Alexa Fluor 594 (cat. no. A-11005/A-11012) were also obtained from Invitrogen; Thermo Fisher Scientific, Inc. Bafilomycin A1 (BAF; 10 nM) and chloroquine (CQ; 20 µ M) were purchased from Sigma-Aldrich; Merck KGaA; sunitinib was from Selleck Chemicals; and Lyso-ER, Lyso-NIR and DAPI were purchased from Abcam.

Techniques: Activation Assay, Cell Culture, Derivative Assay, Incubation, Fluorescence, Staining, Flow Cytometry, Western Blot, Labeling

Journal: International Journal of Oncology

Article Title: Drug resistance-related sunitinib sequestration in autophagolysosomes of endothelial cells

doi: 10.3892/ijo.2019.4924

Figure Lengend Snippet: Reversal of resistance to sunitinib in HMEC-1 cells by autophagy inhibitors. (A-D) HMEC-1 cells were treated with 6 µ M sunitinib for 24 h, either alone or in the presence of 10 nM BAF or 20 µ M CQ for 4 h. DAPI was added to stain the nuclei. (A) Cells were then stained with anti-LAMP and observed under fluorescence microscopy. (B) Treated and non-treated HMEC-1 cells underwent anti-LC3 staining and were observed under fluorescence microscopy. (C) Treated or non-treated HMEC-1 cells underwent Lyso-ER staining for 30 min and were observed under fluorescence microscopy. (D) Treated or non-treated HMEC-1 cells underwent Lyso-NIR staining for 30 min and were (A-D) Magnification, ×63. (E) Survival rates of HMEC-1 cells following treatment with sunitinib with or without BAF. HMEC-1 cells were incubated with or without 6 µ M sunitinib or 10 nM BAF for 48 h. Cells were trypsinized and counted. * P<0.05. (F) Survival rates for HMEC-1 cells following treatment with sunitinib with or without CQ. HMEC-1 cells were incubated with or without 6 µ M sunitinib or 20 µ M CQ for 48 h. Cells were trypsinized and counted. * P<0.05. (G) Survival rates for HMEC 6 µ M cells following treatment with sunitinib with or without BAF or CQ. HMEC 6 µ M cells were incubated with or without 6 µ M sunitinib, 10 nM BAF or 20 µ M CQ for 48 h. Cells were trypsinized and counted. * P<0.05. The results in e, f and g are presented as the mean ± SEM. (H) Steady decrease in sunitinib fluorescent density in cell vesicles following the withdrawal of sunitinib from culture media. HMEC 6 µ M cells were washed and cultured in normal culture medium. The cells were immediately placed into the culture chamber of a time-lapse imaging system. After image zone selection, image recording usually began after 15 min. The images were taken every 12 min and the images at indicated time points are shown. The upper row shows optic microscope images, the middle row shows green fluorescent microscope images and the lower row shows merged photos (magnification, ×40). BAF, bafilomycin A1; CQ, chloroquine; LAMP-1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1A/1B-light chain 3; Suni, sunitinib.

Article Snippet: Anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. 0231-100/LC3-5F10) was obtained from Enzo Life Sciences, Inc.; anti-lysosomal-associated membrane protein 1 (LAMP-1; H4A3; cat. no. sc-20011) was purchased from Santa Cruz Biotechnology, Inc.; anti-sequestosome 1 (SQSTM1)/p62 (cat. no. 5114) was obtained from Cell Signaling Technology, Inc.; horseradish peroxidase-labeled anti-rabbit/mouse IgG antibodies (cat. no. 31460/31430) were obtained from Invitrogen; Thermo Fisher Scientific, Inc.; anti-β-actin (cat. no. A5316) was purchased from Sigma-Aldrich; Merck KGaA; goat anti-mouse IgG and goat anti-rabbit IgG coupled to Alexa Fluor 594 (cat. no. A-11005/A-11012) were also obtained from Invitrogen; Thermo Fisher Scientific, Inc. Bafilomycin A1 (BAF; 10 nM) and chloroquine (CQ; 20 µ M) were purchased from Sigma-Aldrich; Merck KGaA; sunitinib was from Selleck Chemicals; and Lyso-ER, Lyso-NIR and DAPI were purchased from Abcam.

Techniques: Staining, Fluorescence, Microscopy, Incubation, Cell Culture, Imaging, Selection