Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: NanoBRET dilution assay reveals two putative heterodimers, ABCB5β/B6, and ABCB5β/B9. HEK-293T cells were transfected with decreasing amounts of NanoLuc and HaloTag constructs. Transfection started at 1 μg and gradually decreased to 0.125 μg per construct. DMSO refers to the technical negative control to which DMSO is added instead of ligand. Protein pairs serving as negative controls (ABCB2-ABCD1, ABCB3-ABCD1, ABCD1-ABCB5β) ( A ) and a positive control pair (ABCB2-ABCB3) ( B ) were tested alongside ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9 ( C ). Data are presented as mean ± SD. Simple linear regression was used to test whether the amount transfected significantly influenced the NanoBRET ratio ( n = 3). p -values are represented on graphs as ∗∗∗ p < 0.001, ∗∗ p < 0.01, and p > 0.05. For ABCB2-ABCD1 ( R 2 = 0.8719, F (1,10) = 68.07), ABCB3-ABCD1 ( R 2 = 0.6144, F (1,10) = 15.94), ABCB5β-ABCD1 ( R 2 = 0.6661, F (1,10) = 19.95), and ABCB5β-ABCB8 ( R 2 = 0.7526, F (1,10) = 30.41), the relation between both parameters was statistically significant. For ABCB2-ABCB3 ( R 2 = 0.0269, F (1,10) = 0.2767), ABCB5β-ABCB6 ( R 2 = 0.0011, F (1,10) = 0.0113), and ABCB5β-ABCB9 ( R 2 = 0.0204, F (1,10) = 0.2080), changes in the amount of transfected DNA did not influence the NanoBRET ratio. DMSO, dimethyl sulfoxyde; NanoBRET, nanoluciferase-based bioluminescence resonance energy transfer.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Dilution Assay, Transfection, Construct, Negative Control, Positive Control, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Saturation assay confirms specific ABCB5β–ABCB6 and ABCB5β–ABCB9 interactions. A , the concentration of the donor is held constant while the concentration of the acceptor is increased. If the interaction is specific, the signal will reach a plateau where all donors are saturated with acceptors, and luminescence will no longer increase. In the case of a nonspecific interaction, the signal will continue to increase linearly. For each square, the schematic gray halo represents the intensity of the NanoBRET signal observed from low to high. In graphs ( B and C ), the percent of the maximum NanoBRET ratio is plotted against the NanoLuc−HaloTag ratio. B , donor saturation assay for the positive control, ABCB2-ABCB3, and the negative controls, ABCB2-ABCD1, ABCB3-ABCD1, and ABCB5β-ABCD1. C , donor saturation assay for ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9. NanoBRET, nanoluciferase-based bioluminescence resonance energy transfer.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Saturation Assay, Concentration Assay, Positive Control, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Coimmunoprecipitation of ABCB5β-ABCB6 and ABCB5β-ABCB9 demonstrates the presence of these heterodimers in Mel JuSo and UACC-257 cells. The Mel JuSo ( A ) or UACC-257 ( B ) proteins were immunoprecipitated (IP) with either an anti-ABCB5, anti-ABCB6, or anti-ABCB9 antibody. The precipitated proteins were revealed by Western blotting after SDS-PAGE using the corresponding antibody indicated on the right side of each blot. Fifteen micrograms of total proteins from the starting cell lysate were loaded in the first lane, while the total IP eluate was loaded on the gel. ABCB5β was expressed in High-Five insect cells, and total membrane proteins were prepared and loaded on the gel (High5 ABCB5β) as a complementary molecular weight marker when using anti-ABCB5. An isotype control was performed to determine the specificity of the signal obtained in Western blot.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot, SDS Page, Membrane, Molecular Weight, Marker, Control

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Proximity ligation assay confirms the interactions between ABCB5β-ABCB6 and ABCB5β-ABCB9 protein pairs in Mel JuSo and UACC-257. A , confocal images of PLA in Mel JuSo and UACC-257 that stably expressed either a nontargeting shRNA or an ABCB6-specific (or ABCB9-specific) shRNA. The detected PLA signal is in red . Nuclei are stained in blue using DAPI. The scale bar represents 40 μM. After shRNA knockdown, efficiency was determined using reverse transcription-quantitative polymerase chain reaction and Western blots in Mel JuSo ( B ) and UACC-257 ( C ). 18S and tubulin were used as references for reverse transcription-quantitative polymerase chain reaction and Western blots normalization, respectively (n = 2). ImageJ ( https://imagej.net/ij/download.html ) was used to quantify protein expression in the Western blot as well as the average number of dots per cell ( n = 3) ( D ). Scramble and shRNA quantifications are shown along with technical negative controls, including PLA conducted using either one antibody (AB) or none. Data is presented as mean ± SD. In Mel JuSo: ABCB6 scramble (39.89 ± 14.21), ABCB6 shRNA (0.28 ± 0.60), ABCB5 AB control (0.55 ± 0.91), ABCB6 AB control (0 ± 0), ABCB9 scramble (16.89 ± 4.27), ABCB9 shRNA (0.84 ± 1.04), ABCB9 AB control (0 ± 0.01), and no antibody control (0.01 ± 0.05). In UACC-257: ABCB6 scramble (42.16 ± 10.62), ABCB6 shRNA (0.97 ± 1.00), ABCB5 AB control (0.01 ± 0.02), ABCB6 AB control (0.03 ± 0.07), ABCB9 scramble (25.39 ± 6.74), ABCB9 shRNA (1.04 ± 1.16), ABCB9 AB control (0.79 ± 0.58), and no antibody control (0 ± 0). Student’s t test was performed between the scramble and shRNA conditions. Statistical significances are presented on graph as ∗∗∗∗ p < 0.0001. ABCB5β/B6 in Mel JuSo ( t = 9.648, df = 22), ABCB5β/B9 in Mel JuSo ( t = 12,66, df = 22), ABCB5β/B6 in UACC-257 ( t = 13,38, df = 22), ABCB5β/B9 in UACC-257 ( t = 12,34, df = 22). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Proximity Ligation Assay, Stable Transfection, shRNA, Staining, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression of ABCB5β, ABCB6, ABCB5β_P-gp linker_ABCB6, ABCB6_P-gp linker_ABCB5β, ABCB9, ABCB5β_P-gp linker_ABCB9, and ABCB9_P-gp linker_ABCB5β in High-Five insect cells. Total membranes vesicles prepared from High-Five cells infected with baculovirus containing either ABCB5β, ABCB6, ABCB9, ABCB5β_P-gp linker_ABCB6, ABCB6_P-gp linker_ABCB5β, ABCB5β_P-gp linker_ABCB9, ABCB9_P-gp linker_ABCB5β and their corresponding EQ mutants were subjected to SDS-PAGE, followed by Coomassie-blue staining ( A ) or Western blotting with anti-ABCB5, anti-ABCB6, and anti-ABCB9 antibodies ( B ). C , Western blotting with an anti-ABCB1 antibody was performed to assess possible cross reactivity with ABCB5β, ABCB6, and ABCB9 and their chimeric heterodimers. Bands of interest are highlighted by black arrows .

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression of homodimers in High-Five cells and BeFx-sensitive ATPase activity. A , two-dimensional schematic representation of ABCB5β ( purple ), ABCB6 ( orange ), and ABCB9 ( gray ) structure based on CCTOP predictions . ABCB5β has one complete and another partial NBD and one TMD comprised of six transmembrane helices (TMs). The N-terminal NBD (NBD1) is truncated and lacks the conserved Walker A domain. ABCB6 has six transmembrane helices (TM6-TM11) that constitute its TMD1 and five additional transmembrane helices (TM1-TM5) in the N terminus that form the TMD0. ABCB9 has a TMD1 consisting of six transmembrane helices (TM5-10) and a TMD0 made of four transmembrane helices at the N terminus. The location of each EQ mutation is represented by red Xs. B , pixel intensity quantification of the protein bands of interest, after Coomassie-blue staining was performed using ImageJ (n = 3). C , ABCB5β, ABCB6, ABCB9, and their corresponding EQ mutants’ ATPase activities were measured in the presence and absence of BeFx as described in the methods section. On the left , data are shown as ATPase activity in the absence of BeFx (total ATPase activity) and in the presence of BeFx (BeFx-sensitive ATPase activity). On the right , data are represented by subtracting BeFx-resistant ATPase activity from the total ATPase activity (n = 3). In all panels, the names of EQ mutants are shown in red . NBD, nucleotide-binding domain; BeFx, beryllium fluoride.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Mutagenesis, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression level and BeFx-sensitive ATPase activity of heterodimers. A , two-dimensional schematic representation of the ABCB5β/B6 and ABCB5β/B9 heterodimers after fusion with the P-gp flexible linker based on CCTOP predictions . The P-gp linker is shown in green and highlighted with a green arrow ; ABCB5β is represented in purple , ABCB6 in orange , and ABCB9 in gray . The ABCB5β_P-gp linker_ABCB6 has 16 transmembrane helices. The ABCB6_P-gp linker_ABCB5β has 17 transmembrane helices and the ABCB5β_P-gp linker_ABCB9 and ABCB9_P-gp linker_ABCB5β both have 16 transmembrane helices. The location of EQ mutations is represented by red Xs. B , the Coomassie-blue stained gel pixel intensity of the bands of interest were quantified using ImageJ (n = 3). C , the graph on the left shows the ATPase activity for all heterodimer chimeras and their corresponding EQ mutants measured in both the absence and presence of BeFx as described in the method section, total ATPase activity, and BeFx-sensitive ATPase activity, respectively. On the right , data represent BeFx-sensitive ATPase activity, which is calculated by subtracting BeFx ATPase activity from the total ATPase activity (n = 3). In all panels, the names of EQ mutants are shown in red . BeFx, beryllium fluoride.

Article Snippet: Five milligrams of total protein (from either HEK93T, Mel Juso, or UACC-257) in lysis buffer (120 mM NaCl, 50 mM Tris, NP40 0.5%, 5 mM EDTA, 1× protease inhibitor) were incubated on a rotating wheel at 4 °C for 4 h with 1 μg of antibody: ABCB5 (600-401-A775, Rockland Immunochemicals), ABCB6 (G-10, Santa Cruz Biotechnology), ABCB9 (A-8, Santa Cruz Biotechnology), IgG Mouse (sc-2025, Santa Cruz Biotechnology, Dalla), and IgG Rabbit (sc-3888, Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Staining

Journal: Advanced healthcare materials

Article Title: Hydrogel microtumor arrays to evaluate nanotherapeutics

doi: 10.1002/adhm.202201696

Figure Lengend Snippet: Mechanical cues prime a population of cells with increase stemness which increase uptake of Cy5-PFC3 (A) Representative image of fluorescence intensity quantitation in B16F0 cells (Image J). Cells outlined in red considered ‘perimeter’ and yellow considered ‘centre’. Heatmap of nanoparticle intensity after five days culture (n=10). (B) Scatter plot of fluorescence intensity of B16F0 cells localised at the perimeter and the centre of a pattern cultured for five days after 1 h treatment with Cy5-PFC3. Significant difference in uptake between cells in perimeter and centre (p = 0.0002, n = 12) and ABCB5 expression (p = 0.0079, n = 12). (C) Scatter plot of fluorescence intensity of B16F0 and B16F10 cells in spiral patterns cultured on 10 kPa polyacrylamide substrate. Error bars denote ± standard deviation. Significant difference in Cy5-PFC3 uptake between B16F0 1 day and 5 days (p = 0.0005, n = 15), B16F0 and B16F10 at 5 days (p < 0.0001, n = 15) and ABCB5 expression between B16F0 1 day and 5 day (p < 0.0001, n = 15), B16F0 and B16F10 at 5 days (p < 0.0174, n = 20)

Article Snippet: Gels were then incubated with the primary antibody (rabbit polyclonal anti-ABCB5, Novus Biologicals) diluted at 1:500 in 1% BSA in PBS for 1h at room temperature.

Techniques: Fluorescence, Quantitation Assay, Cell Culture, Expressing, Standard Deviation

Journal: Advanced healthcare materials

Article Title: Hydrogel microtumor arrays to evaluate nanotherapeutics

doi: 10.1002/adhm.202201696

Figure Lengend Snippet: Shape induced and innate cellular ABCB5 expression increases nanoparticle uptake in murine melanoma cells. (Scale bar = 100 μm) (A) Representative fluorescence images of a B16F0 one day culture, B16F0 five day culture, B16F10 one day culture, and B16F10 five day culture. Fluorescence channels present Cy5-PFC3 nanoparticle (Red), ABCB5 putative cancer stem cell marker (Alexa Fluor 555, Orange) and cancer cell nuclei (DAPI) on spiral patterns. (B) Quantification of mean Cy5-PFC3 and ABCB5 stem cell marker intensity in B16F0 (n = 10) and B16F10 (n = 10) cells proliferated under spiral geometry for one day or five days with standard deviation shown with error bars. ****: P ≤ 0.0001, ***: P ≤0.001

Article Snippet: Gels were then incubated with the primary antibody (rabbit polyclonal anti-ABCB5, Novus Biologicals) diluted at 1:500 in 1% BSA in PBS for 1h at room temperature.

Techniques: Expressing, Fluorescence, Marker, Standard Deviation

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: WM-266-4 cells were surface-labelled with the ABCB5-Ab Rock antibody and analyzed by flow cytometry. ABCB5 + cells (right contour plot) were gated on viable cells (DAPI-negative) according to the isotype control (left contour plot). Inserts (dot plots) display the gating of the positive cells ( A ). WM-266-4 cells were treated with a siRNA designed to target ABCB5 (si-ABCB5). After 72 h, the cells were analyzed for their ABCB5 mRNA content and ABCB5 surface expression. The left and right histograms show respectively the relative expression of ABCB5 mRNA normalized to the ABCB5 mRNA in cells treated with a control siRNA (si-ctrl), and the percentage of ABCB5 + cells among total cells (n = 3). The corresponding contour plots are shown ( B ). Different melanoma cell lines were analyzed for their ABCB5 surface expression ( C ) or their ABCB5 mRNA content ( D ) (n = 3).

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: WM-266-4 cells (5×10 6 cells) were injected subcutaneously in Swiss nude mice. Fourteen days later, mice were treated by repeated i.p. injections of either temozolomide (80 mg/kg) or vehicle following the schedule indicated by the black arrows. The tumoral volumes were monitored and the means of measured volumes respectively for temozolomide-treated and vehicle treated tumors are as follows: 180 mm 3 and 175 mm 3 at day 14; 264 mm 3 and 295 mm 3 at day 16; 178 mm 3 and 444 mm 3 at day 18; 84 mm 3 and 699 mm 3 at day 21. ( A ). 24 h after the injections at days 16 and 21 (d17 and d22), tumors were recovered, dissociated and the cell suspensions were searched for the presence of human ABCB5 + cells by flow cytometry ( B ) (medians are represented as black lines). Tumors recovered at day 17 were analyzed by immunohistochemistry for their ABCB5 expression ( C ).

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Injection, Flow Cytometry, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: Skin metastases specimens from respectively 8 untreated and 7 treated patients were analyzed by immunohistochemistry for their ABCB5 protein expression. The ABCB5 staining intensity was ranked in four arbitrary classes according to the intensity and the extent of the labelling. Representative staining of two levels of intensity (left panel: isotypic control) (A). Repartition of the specimens in the different classes (B). The two groups of specimens (untreated versus treated) have been compared with the non parametric Kruskall Wallis test (p<0.30).

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Immunohistochemistry, Expressing, Staining

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: WM-266-4 ( A,D,G ), G-361 ( B,E,H ) and SK-MEL-28 cells ( C,F,I ) were treated at the indicated concentrations of dacarbazine ( A–C ), vemurafenib ( D–F ) and doxorubicin ( G–I ). After 72 h, the total viable cells were numbered using an automated cell viability analyzer. The percentages of viable ABCB5-expressing cells (among viable cells gated on DAPI-negative cells) were analyzed by flow cytometry. The numbers of total cells (white symbols) are reported as percentages of the number of cells in the untreated control sample. The numbers of viable ABCB5 + cells (black symbols) were calculated from the total cell numbers and ABCB5 + cells ratio, and reported as percentages of the viable ABCB5 + cells number in the control sample. ABCB5 + cells represent respectively 3%, 3.5% and 5% of the total cells in the WM-266-4, G-361 and SK-MEL-28 cell lines.

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: WM-266-4 cells were treated with dacarbazine, doxorubicin or vehicle (NT) for 72 h. Cycloheximide ( A–B ) or brefeldin A ( C–D ) were added respectively 24 h and 4 h before the treatment end-point. Cells were labelled for ABCB5 and analyzed by flow cytometry. The means of fluorescence intensity of the ABCB5 + cells from three independent experiments were reported in A and C . Fluorescence intensity histograms of representative experiments are shown in the . The number of ABCB5 + cells was reported in B and D as a percentage of the ABCB5 + cell number in the vehicle-treated sample.

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

doi: 10.1371/journal.pone.0036762

Figure Lengend Snippet: WM-266-4 cells were treated for 72 h with the indicated concentrations of doxorubicin, dacarbazine, vemurafenib, gemcitabine or with vehicle (NT) and ABCB5 expression was analyzed by Western blot. Band intensities were quantified and variations are indicated as fold increases in treated versus untreated samples ( A ). WM-266-4 cells were treated with various drugs at their EC50 for 72 h. The percentages of positive cells among surviving cells were measured by cell surface labelling and flow cytometry analysis for ABCB5 ( B ) or ABCB1 ( C ). The relative mRNA expression of ABCB5, ABCB1, ABCC1, ABCG2 and HMBS as the house-keeping gene was measured by Q-PCR (see also ) and the amplified products were run on agarose gel after 29 cycles except for ABCB1 (32 cycles) ( D ).

Article Snippet: Slides were incubated with a rabbit polyclonal anti-ABCB5 antibody ABCB5-Ab Sigma (Sigma) followed by a secondary Horse Radish Peroxidase-rabbit antibody and visualized using a colorimetric method (Envision kit, Dako, Glostrup, Denmark).

Techniques: Expressing, Western Blot, Flow Cytometry, Amplification, Agarose Gel Electrophoresis

Journal: Stem Cell Research & Therapy

Article Title: Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model

doi: 10.1186/s13287-017-0707-y

Figure Lengend Snippet: Tissue explant and single cell-suspension cultures produced more LESCs and fewer stromal cells. a , b ABCB5 and p63α positivity in rabbit limbus, with negativity in central cornea. Arrows point to the ABCB5 + and p63α + LESCs in the limbus. c , d Expressions of proposed LESC markers (p63α and ABCB5) and stromal cell marker (α-SMA) in P1 and P2 LESCs from the three cultures. Data were given as mean ± SD from three independent experiments. One-way ANOVA analysis: * P < 0.05; ** P < 0.01; *** P < 0.001. e Flow cytometric staining (gating based on control staining) for p63α and ABCB5 of LESCs (P1) from the three cultures. Scale bar, 50 μm. ABCB5 sub-family B, member 5, α-SMA alpha-smooth muscle actin

Article Snippet: The following antibodies were used: rabbit anti-p63α monoclonal antibody (ab124762; Abcam), mouse anti-ABCB5 monoclonal antibody (ab140667; Abcam), rabbit anti-α-SMA polyclonal antibody (BM0002; Boster), mouse anti-Ki67 monoclonal antibody (550609; BD BioSciences), goat anti-CK12 polyclonal antibody (sc-17098; Santa Cruz), rabbit anti-CK12 monoclonal antibody (ab185627; Abcam), mouse anti-CK7 antibody (ab9021; Abcam), mouse anti-BrdU monoclonal antibody (ab1893; Abcam), rabbit anti-Claudin-1 polyclonal antibody (ab15098; Abcam), mouse anti-CD68 monoclonal antibody (MCA341R; AbD Serotec), and mouse anti-CD31 monoclonal antibody (ab9498; Abcam).

Techniques: Suspension, Produced, Marker, Staining, Control