1. Cell Cycle Phase-Specific Drug Resistance as an Escape Mechanism of Melanoma Cells 
Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Goldie Y.L. Lui, Danae M. Sharp, Brian Gabrielli, Wolfgang Weninger, Nikolas K. Haass 
Journal of Investigative Dermatology 
Volume 136, Issue 7, Pages (July 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Bortezomib induces dose-dependent G2 arrest of melanoma cells. (a) Flow cytometric cell cycle profile of FUCCI-C8161 cells cultured in two dimensions treated with vehicle (control) or 10 nM bortezomib for 24 hours. DNA content histograms are overlaid with the FUCCI distribution. Graphs are representative of n = 3 independent experiments. (b) Fluorescence microscopic image analysis of % G1, early S, and S/G2/M FUCCI-C8161 cells cultured in two dimensions after 24 hours of bortezomib treatment. Representative of n = 2 independent experiments. (c) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay of C8161 cells treated with bortezomib for 48 hours at the indicated concentrations. n = 3. Values are given as mean ± SEM. All samples were compared to the control sample to determine statistical differences. ***P < 0.001; ****P < DAPI, 4′,6-diamidino-2-phenylindole; FUCCI, fluorescent ubiquitination-based cell cycle indicator; ns, not significant; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Bortezomib induces G2 and G1-arrest. (a) Confocal extended focus images of collagen-embedded FUCCI-C8161 spheroids treated with 10 nM bortezomib for indicated times. Top slices of the z-stack were removed to reveal the red, G1-arrested spheroid core. Images are representative of n = 3 experiments. Gray line indicates spheroid edge as seen in bright field. Insets shows untreated control. Bar = 200 μm. (b) Flow cytometric cell cycle profiles of live cells of FUCCI-C8161 spheroids treated with vehicle (control) or 10 nM bortezomib for indicated periods. DNA content histogram is overlaid with the FUCCI distribution. Graphs are representative of n = 3 independent experiments. (c) Flow cytometric analysis of live FUCCI-C8161 spheroids (left) or two-dimensional cultured FUCCI cells (right) after 24-, 48-, and 72-hour treatment with 10 nM bortezomib. n = 2–4 experiments. Values are given as mean ± SEM. BTZ, bortezomib; DAPI, 4′,6-diamidino-2-phenylindole; FUCCI, fluorescent ubiquitination-based cell cycle indicator; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Bortezomib induces apoptosis preferentially of G2-phase cells. (a) Flow cytometric quantification of % cell death in C8161 cells cultured in two (2D) or three dimensions (3D) and treated with vehicle (control) or 10 nM bortezomib for the indicated time. n = 2–4. Values are given as mean ± SEM. (b) Percent cells (2D) in G1 versus S/G2/M phase at death assessed by time-lapse image analysis. Error bars = mean ± SEM. n = 2–3. Cell death was determined by cell morphology. (c) G1 versus S/G2/M phase at time of bortezomib-induced death (2D) assessed by time-lapse image analysis. Cell death was ascertained by cell morphology. Error bars = mean ± SEM. Data pooled from n = 2–3 experiments. (d) The 2D-cultured FUCCI-C8161 cells were treated with 10 nM bortezomib for the indicated times. Percentage of annexin V-positive cells in G1 or S/G2/M phase was quantified by flow cytometry. Error bars = mean ± SEM. n = 3. (e) FUCCI-1205Lu cells treated for 24 hours with 10 nM bortezomib were sorted into G1-phase or S/G2/M-phase live cell fractions, or were left unsorted before immunoblotting. Blots are representative of n = 3 experiments. Black line indicates separate blots. *P < 0.05; ****P < SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
Pharmacologically induced G1 arrest inhibits bortezomib and temozolomide-induced cytotoxicity. (a) Time line demonstrating the drug treatment schedule. Flow cytometric quantification of % cell death in (b) WM164, (c) 1205Lu, and (d) C8161 cells cultured in two dimensions (2D) and treated with vehicle (DMSO), 10 nM BTZ, 10 μM U0126 (1μM for WM164), 1 μM PLX4032 (0.1 μM for WM164), or the indicated combinations. BTZ was added after 24 hours, for a total of 48 hours, whereas the other treatments were applied for the total 72 hours. Error bars = mean ± SEM. n = 3–5. Samples were compared to DMSO+BTZ to determine statistical differences. (e) Confocal extended focus images of FUCCI-C8161 spheroids treated with vehicle (DMSO), 15 nM BTZ alone, or combined with 10 μM U0126 or 1 μM PLX4032. BTZ was only added after 24 hours, for a total of 24 hours, whereas the other treatments were applied for the total 48 hours. (f) Flow cytometric quantification of % cell death in melanoma cells cultured in 2D. Cell line: WM164; treatment: vehicle (DMSO), 10 nM BTZ, 0.1 μM PLX4032, or PLX + BTZ for 48 hours. Drugs were added simultaneously. Samples were compared to DMSO+BTZ to determine statistical differences. Error bars = mean ± SEM. n = 3. (g) As in f with the following variables: cell line: WM164; treatment: 10 μM U0126, 0.1 μM PLX4032, or combination. U0126 was added after 24 hours. PLX4032 was present for the entire 72 hours. (h) Image analysis of the % G1, early S, and S/G2/M FUCCI-C8161 cells cultured in 2D after 24-hour treatment with 10 μM TMZ. n = 3. Values are given as mean ± SEM. (i) As in f with the following variables: cell line: C8161; treatment: 10 μM U0126, 40 μM TMZ, or combination. TMZ was added after 24 hours. U0126 was present for the entire 72 hours. (j) As in f with the following variables: cell line: C8161; treatment: 10 μM TMZ, 60–80 μM U0126, or combination. U0126 was added after 24 hours. TMZ was present for the entire 72 hours. (k) As in f with the following variables: cell line: C8161; treatment: 10 μM TMZ, 10 nM BTZ, or combination. BTZ was added after 24 hours. TMZ was present for the entire 72 hours. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < BTZ, bortezomib; ns, not significant; SEM, standard error of the mean; TMZ, temozolomide.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Environmentally induced G1 arrest inhibits bortezomib-induced cell death but enhances MAPK inhibitor cytotoxicity. (a) Image analysis of the % G1, early S, and S/G2/M phase WM164 cells cultured in two dimensions (2D) in normoxia with normal medium or after 24 hours of hypoxia and serum-starvation. Values are given as mean ± SEM. n = 4. (b) Image analysis of the % red G1, early S, and S/G2/M phase C8161 cells cultured in 2D in control or in confluent/starved wells after 48 hours. Values are given as mean ± SEM. n = 3. (c) FUCCI-WM164 cells cultured in 2D were pretreated with either hypoxia and serum-starvation for 24 hours, or control normoxia and full serum conditions. Flow quantification of the % dead cells was performed after 48-hour treatment with 15 nM bortezomib or vehicle control. Values are given as mean ± SEM. n = 4. (d) FUCCI-C8161 cells cultured in 2D were pretreated with either subconfluency with normal medium or confluency with serum-starvation for 48 hours. Flow quantification of the % dead cells was performed after an additional treatment with 10 nM bortezomib (BTZ), 40 μM TMZ, or vehicle control for 48 hours. Values are given as mean ± SEM. n = 3–4. (e) As in c but treatment with 20 μM U0126. Values are given as mean ± SEM. n = 3. (f) As in d but treatment with 60–80 μM U0126. Values are given as mean ± SEM. n = 3. (g) WM164 cells were cultured in 2D in normoxia with normal medium, or after hypoxia and serum-starvation for 24 or 48 hours. Western blotting was then performed. Blots are representative of n = 3 independent experiments. *P < 0.05; **P < 0.01; ***P < ns, not significant; SEM, standard error of the mean; TMZ, temozolomide.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions