1. Volume 29, Issue 6, Pages 805-819 (June 2016)
Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance 
Michaël Cerezo, Abdelali Lehraiki, Antoine Millet, Florian Rouaud, Magali Plaisant, Emilie Jaune, Thomas Botton, Cyril Ronco, Patricia Abbe, Hella Amdouni, Thierry Passeron, Veronique Hofman, Baharia Mograbi, Anne-Sophie Dabert-Gay, Delphine Debayle, Damien Alcor, Nabil Rabhi, Jean-Sébastien Annicotte, Laurent Héliot, Mariano Gonzalez-Pisfil, Caroline Robert, Solange Moréra, Armelle Vigouroux, Philippe Gual, Maruf M.U. Ali, Corine Bertolotto, Paul Hofman, Robert Ballotti, Rachid Benhida, Stéphane Rocchi 
Cancer Cell 
Volume 29, Issue 6, Pages (June 2016)
DOI: /j.ccell
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2. Cancer Cell 2016 29, 805-819DOI: (10.1016/j.ccell.2016.04.013)
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3. Figure 1 HA15 Exerts a Deleterious Effect on Melanoma Cell Viability
(A) Chemical structure of troglitazone and HA15.
(B) The melanoma cell line A375 was treated with 10 μM HA15. At the indicated times, viable cells were counted using the trypan blue dye exclusion method.
(C) A375 melanoma cells were treated with indicated concentrations of HA15. After 24 hr, viable cells were counted using the trypan blue dye exclusion method.
(D) Indicated melanoma cell lines were treated with 10 μM HA15. After 48 hr, viable cells were counted using the trypan blue dye exclusion method.
(E) Human melanoma cells freshly isolated from tumors were treated for 48 hr with 10 μM HA15. Cell viability was estimated by trypan blue staining.
(F) Primary human melanocytes and fibroblasts were treated for 48 hr with 10 μM and 100 μM HA15. Cell viability was estimated by trypan blue staining.
For (B–F), the results are expressed as percentages of the control and data are means ± SD of three independent experiments performed in triplicate. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S1.
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4. Figure 2 HA15 Induces an Early ER Stress
(A) Gene set enrichment analysis (GSEA) result showing that the gene set related to unfolded protein response scores was enriched in HA15-treated melanoma cells (top) and heatmaps for the gene sets of unfolded protein response in melanoma cells treated for 6 hr with 10 μM HA15 (bottom).
(B) Lysates from A375 melanoma cells exposed for the indicated durations to HA15 were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown.
(C) XBP1 mRNA splicing was detected with RT-PCR. GAPDH was used to confirm the quality and quantity of RNA.
(D) Electron microscopy images presenting the ultrastructure in A375 melanoma cells treated for 48 hr with DMSO (representative control) or 10 μM HA15.
(E) Immunofluorescence pictures of A375 melanoma cells treated with 10 μM HA15 for 6 hr. ER was labeled with an ER Tracker and the HA15 visualized after excitation at 405 nm.
(F) Lysates from indicated melanoma cells exposed to 10 μM HA15 for the indicated duration were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown.
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5. Figure 3 BiP is a Target of HA15
(A) Chemical structure of HA15-biotin and AM406-biotin.
(B) Pull-down of recombinant IRE1α, ATF6, BiP, or HSP70 with HA15-biotin or AM406-biotin. One representative experiment of three is shown.
(C) HA15-biotin, biotin, and AM406-biotin were incubated with lysates of A375 melanoma cells and precipitated using streptavidin beads. Western blotting was performed with indicated antibodies. One representative experiment of three is shown.
(D) PERK, IRE1α, and ATF6 were immunoprecipitated overnight from A375 lysates in the presence of DMSO or HA15. Western blotting was performed with indicated antibodies. One representative experiment of three is shown.
(E) Immunofluorescence pictures of A375 melanoma cells treated with DSMO or 10 μM HA15 for 6 hr. ER was labeled with ER Tracker, BiP was labeled with antibody, and HA15 was visualized after an excitation at 405 nm.
(F) BiP activity measurement on A375 melanoma cells treated with HA15, honokiol (HNK), or DMSO. The results are expressed as percentages of control (DMSO). Data are means ± SD of three independent experiments performed in triplicate. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figures S2 and S3; Table S1.
Cancer Cell  , DOI: ( /j.ccell )
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6. Figure 4 HA15 Induces Concomitant Autophagy and Apoptosis
(A) Electron microscopy images presenting the ultrastructure in melanoma cells treated for 48 hr with DMSO (representative control) or 10 μM HA15.
(B) Cells treated for indicated times with 10 μM HA15 were detached and their DNA contents were measured by flow cytometry. The results are expressed as percentages of the control. Data are means ± SD of three independent experiments performed in triplicate. ∗∗∗p <
(C) A375 melanoma cells were treated for 48 hr with increasing concentrations of HA15. Cell lysates were separated by SDS-PAGE and analyzed by western blotting using the indicated antibody. β-Actin or HSP60 was used as a loading control. One representative experiment of three is shown.
(D) A375 melanoma cell lines were treated with 10 μM HA15 for the indicated periods of time and their lysates were analyzed by western blotting using the indicated antibodies. HSP90 were used as a loading control. One representative experiment of three is shown.
(E) Immunofluorescence pictures of A375 melanoma cells treated with DMSO or 10 μM HA15 for 24 hr. LC3B was labeled with antibody (green) and DNA was visualized with DAPI (blue). Quantifications of LC3B fluorescence intensity are presented in a.u. and represent means ± SD of three independent fields. ∗∗∗p < 0.001.
(F) A375 melanoma cells were transfected with siRNA against CHOP (siCHOP) or siRNA control (siCtl). 24 hr after transfection, cells were treated with HA15 at the indicated concentrations or DMSO for 48 hr. Viability of the cells was estimated by trypan blue staining. The results are expressed as percentages of the control. Data are means ± SD of three independent experiments performed in triplicate. ∗∗∗p < In parallel, cells were lysed and analyzed by western blotting using the indicated antibodies. HSP90 was used as a loading control. One representative experiment of three is shown.
(G) A375 melanoma cells were transfected with a siRNA against LC3 (siLC3) or siRNA control (siCtl) and treated with QVD or DMSO. 24 hr after transfection, cells were treated with 10 μM HA15 or DMSO for 48 hr. Viability of the cells was estimated by trypan blue staining. The results are expressed as percentages of the control. Data are means ± SD of three independent experiments performed in triplicate. ∗∗p < 0.01; ∗∗∗p < 0.001; #p < In parallel, cells were lysed and analyzed by western blotting using the indicated antibodies. HSP60 was used as a loading control. One representative experiment of three is shown.
(H) Normal human fibroblasts (NHF), normal human melanocytes (NHM), and A375 melanoma cells were treated for 48 hr with HA15 (10 μM). Cell lysates were separated by SDS-PAGE and analyzed by western blotting using the indicated antibody. HSP60 was used as a loading control. One representative experiment of three is shown.
See also Figure S4.
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7. Figure 5 HA15 Inhibits Melanoma Tumor Development in Mice
(A) Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with 1.5 × 106 A375 melanoma cells. After 7 days, mice (n = 6 in each group) were treated with PLX4032 or HA15 (0.7 mg/mouse/day) or vehicle. The tumor growth curves were determined by measuring the tumor volume. The bars indicate the mean ± SD. ∗∗∗p <
(B) Mice at the end of the experiment show in (A) were euthanized and tumors were weighed. The bars indicate the mean ± SD. ∗∗∗p <
(C) Evolution of the mouse weight during the xenograft experiment. The bars indicate the mean ± SD.
(D) Liver weight of mice at the end of the experiment show in (A). The bars indicate the mean ± SD.
(E) Frozen sections of tumors from mice treated with vehicle, HA15, or PLX were fixed and stained using CHOP antibody (red), DAPI (blue), or using TUNEL assay (green). The experiment was repeated on sections from three mice per condition. Quantifications of TUNEL and CHOP fluorescence intensity are presented in a.u. and represent means ± SD of three independent fields. ∗p < 0.05.
(F) Frozen sections of tumors from mice treated with vehicle, HA15 or PLX were fixed and stained using LC3 antibody (green) or DAPI (blue). Representative sections are shown. The experiment was repeated on sections from three mice per condition. Quantifications of LC3 fluorescence intensity are presented in a.u. and represent means ± SD of three independent fields. ∗p < 0.05.
(G) Representative photomicrographs of liver sections stained with H&E of mice treated with vehicle, HA15, or PLX4032.
(H) Quantifications of transaminases (AST/ALT) of mice treated with vehicle, HA15, or PLX4032 are means ± SD of six different sera.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 6
HA15 Induces Death of Melanoma Cells that Are Resistant to BRAF Inhibitors
(A) Indicated melanoma cells sensitive (S) or resistant (R) to PLX4032 were treated with 10 μM HA15, 10 μM PLX4032, or DMSO for 48 hr. Cell viability was estimated by trypan blue staining. The results are expressed as percentages of the control. Data are means ± SD of three independent experiments performed in triplicate. ∗∗∗p < In parallel, cells were lysed and analyzed by western blotting using the indicated antibodies. HSP60 was used as a loading control. One representative experiment of three is shown.
(B) Human melanoma cells freshly isolated from tumor resistant to PLX4032 (right) or dabrafenib (left) were treated with 10 μM HA15, 10 μM dabrafenib (Dabra), 10 μM PLX4032, or DMSO for 48 hr. Cell viability was estimated by trypan blue staining. The results are expressed as percentages of the control. Data are means ± SD of three independent experiments performed in triplicate. ∗∗p < 0.01; ∗∗∗p < In parallel, cells were lysed and analyzed by western blotting using the indicated antibodies. HSP60 was used as a loading control. One representative experiment of three is shown.
(C) Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with A375 melanoma cells resistant to PLX4032 and were treated 7 days later with PLX4032 and HA15 (0.7 mg/mouse/day) or vehicle (n = 6 in each group). The tumor growth curves were determined by measuring the tumor volume. The bars indicate the means ± SD. ∗∗∗p <
(D) Mice were euthanized and weighed. The bars indicate the means ± SD. ∗∗∗p <
(E) Frozen sections of tumors from mice treated with vehicle, HA15, or PLX were fixed and stained using CHOP antibody (red), DAPI (blue), or using TUNEL assay (green). Slides were examined with a Zeiss fluorescence microscope. Representative sections are shown. The experiment was repeated on sections from three mice per condition. Quantifications of TUNEL and CHOP fluorescence intensity are presented in a.u. and represent means ± SD of three independent fields. ∗p < 0.05.
(F) Frozen sections of tumors from mice treated with vehicle, HA15, or PLX were fixed and stained using LC3B antibody (green) or DAPI (blue). Slides were examined with a Zeiss fluorescence microscope. Representative sections are shown. The experiment was repeated on sections from three mice per condition. Quantifications of LC3B fluorescence intensity are presented in a.u. and represent means ± SD of three independent fields. ∗p < 0.05.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 7 HA15 Induces Death of Cell Lines Derived from Other Tumors
(A) Various cancer cell lines were treated with 10 μM HA15. After 48 hr, viable cells were counted using the trypan blue dye exclusion method. The bars indicate the mean ± SD. ∗∗∗p <
(B) Human chronic myelogenous leukemia cell lines K562 and ImaR were treated with 10 μM HA15. After 48 hr, viable cells were counted using the trypan blue dye exclusion method. The bars indicate the mean ± SD. ∗∗p < 0.01 ∗∗∗p <
(C) Indicated cells were exposed during 48 hr to 10 μM HA15. Lysates were analyzed by western blotting using the indicated antibodies. One representative experiment of three is show.
Cancer Cell  , DOI: ( /j.ccell )
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