Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: Active integrin β3 distribution and colocalization with pFAK 397 within focal adhesions are influenced by ABCA1 activity. A and C Representative confocal images of scrambled and ABCA1 KO cells ( A ) or DMSO- and probucol-treated cells ( C ) stained with an antibody recognizing pFAK 397 followed by a secondary antibody conjugated with Alexa Fluor 488 (green) and antibody against active integrin β3 followed by secondary antibody conjugated with Alexa Fluor 647 (red). Scale bars represent 20 µm. B and D Magnified view of scrambled (ROI1) and ABCA1 KO (ROI2) cells ( B ) from ( A ) or of DMSO- (ROI3) and probucol-treated (ROI4) cells ( D ) from ( C ). White arrowheads point at pFAK 397 -rich active integrin β3 clusters. Scale bars represent 20 µm. E , F Quantification of the percentage of the cells with active integrin β3 clusters enriched in pFAK 397 of scrambled and ABCA1 KO cells ( E ) or DMSO- and probucol-treated cells ( F ) (n = 30 from three independent experiments, unpaired Student t-test). G , H Quantification of clusters number of active integrin β3 of scrambled and ABCA1 KO cells ( G ) from ( A ) or DMSO- and probucol-treated cells ( H ) from ( C ) (n = 30 from three independent experiments, unpaired Student t-test). I , J Quantification of the clusters size of active integrin β3 of scrambled and ABCA1 KO cells ( I ) from ( A ) or DMSO- and probucol-treated cells ( J ) from ( C ) (n = 30 from three independent experiments, unpaired Student t-test). ROI region of interest. Data are mean ± SD. **p ≤ 0.01 and ****p ≤ 0.0001

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Activity Assay, Staining

Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: ABCA1 regulates Hs294T PM lateral organization, fluidity and order to promote the formation of fully active FAs. A , B Representative FLIM images of cellular PM, which were obtained after staining of scrambled and ABCA1 KO cells ( A ) or DMSO- and probucol-treated cells ( B ) with the Di-4 probe. The code color scale represents the average lifetime of Di-4 with 2.9 ns as a minimum (blue) and 4.5 ns as maximum (red). Scale bars reprensent 20 µm. C , D Quantitative analysis of Di-4 mean lifetime for scrambled and ABCA1 KO cells ( C ) and for DMSO- and probucol-treated cells ( D ) (n = 30 from three independent experiments, unpaired Student t-test). E , F Distribution represented as % of Di-4 intensity lifetime separated into two segments: Di-4 intensity with a lifetime between 3 and 4 ns [3, 4] and between 4 and 5 ns [4, 5] for scrambled and ABCA1 KO cells ( E ) and for DMSO- and probucol-treated cells ( F ) (n = 30 from three independent experiments, unpaired Student t-tests). G , H Representative confocal images of scrambled and ABCA1 KO cells ( G ) or DMSO- and probucol-treated ( H ) cells loaded or not with cholesterol using a methyl-β-cyclodextrin-cholesterol complex (Chol) for 30 min. Upon fixation, the cells were stained with an antibody recognizing pFAK 397 followed by a secondary antibody conjugated with Alexa Fluor 488 (green) and an antibody against active integrin β3 followed by a secondary antibody conjugated with Alexa Fluor 647 (red). The region of interest (blue squares) of the cells was magnified (ROI). White arrowheads point at pFAK 397 -rich active integrin β3 clusters. Scale bars represent 20 µm. I , J Quantification of clusters number of active integrin β3 of scrambled and ABCA1 KO cells ( I ) or DMSO- and probucol-treated cells ( J ) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). K , L Quantification of the size of clusters with active integrin β3 of scrambled and ABCA1 KO cells ( K ) or DMSO- and probucol-treated cells ( L ) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). Data are mean ± SD. ns non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Staining

Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: Model of the ABCA1 activity influence on Hs294T cells motility and ECM degradation processes. A (1) ABCA1 activity influences the cholesterol distribution within Hs294T PM, creating a heterogeneous lipid packing where lipids and membrane proteins can diffuse actively. ( 2 ) This specific PM lateral organization leads to the formation of integrin αVβ3 clusters that elicit activation of integrin β3 and trigger stable docking of phosphorylated FAK at the FAs and migration of the cells. (3) The specific PM lateral orsganization also influences the formation of active invadopodia which in turn increases the ECM degradation. (4) The rise in migration and the ECM digestion finally enhances the melanoma cells’ invasion potential. B (1) Loss of ABCA1 expression or activity triggers the accumulation of cholesterol within the PM and its excess is transformed into esterified cholesterol in lipid droplets. The PM fluidity decreases as the lipid packing rises, making the PM more homogeneous. (2) The decrease of PM fluidity destabilizes the integrin αVβ3 (with active integrin β3) clusters formation and the retaining of phosphorylated FAK at FAs, thus inhibiting the motility of the cells. (3) Accumulation of cholesterol and alteration of the PM lateral organization also inhibit the active invadopodia formation, partially preventing the ECM degradation. (4) Combined decrease in cells migration and ECM degradation lowers melanoma cells’ metastatic potential

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Activity Assay, Activation Assay, Migration, Expressing, Transformation Assay

Journal: Bio-protocol

Article Title: Measurement of redox states of the β3 integrin disulfide bonds by Mass Spectrometry

doi: 10.21769/BioProtoc.3156

Figure Lengend Snippet: Monoisotopic m/z ratios for the β3 integrin YCNCTTR peptide alkylated with 12 C-IPA and 13 C-IPA

Article Snippet: Ziptip ® C18 pipette tip (Millipore, catalog number: ZTC18S96) 1.5 ml tubes Natural human αIIbβ3 protein purified from human platelets (Abcam, catalog number: ab95130) Anti-integrin β3 (GPIIIa, CD61) PSI domain [AP-3] antibody (Kerafast, catalog number: EBW106) 2-iodo-N-phenylacetamide ( 12 C-IPA) (Cambridge isotopes, catalog number: ULM-8131-PK) 13 C-2-iodo-N-phenylacetamide ( 13 C-IPA) (Cambridge isotopes, catalog number: CLM-8130-0.1) Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418) NuPAGE TM 4x LDS sample loading buffer (Thermo Fisher Scientific, catalog number: NP0007) NuPAGE TM 4-12% Bis-Tris gel 1.5 mm, 10-well (Thermo Fisher Scientific, catalog number: NP0335BOX) NuPAGE TM MOPS running buffer 20x (Thermo Fisher Scientific, catalog number: NP0001) Brilliant Blue G solution (Sigma, catalog number: B8522-1EA) Ammonium bicarbonate (Sigma, catalog number: A6141) Dithiothreitol (DTT) (Sigma, catalog number: D0632) Formic acid LC-MS grade (Thermo Fisher Scientific, catalog number: 28905) Trifluoroacetic acid (Sigma, catalog number: 302031) Formic acid (Sigma, catalog number: F0507) Acetonitrile HPLC grade (Fisher Chemical, catalog number: 10407440) PNGase F Proteomics grade enzyme (Sigma, catalog number: P7367) Sequencing grade bovine chymotrypsin (Roche, catalog number: 11418467001) Trypsin Gold, Mass spectrometry grade (Promega, catalog number: V5280) HCl Acetic acid Calcium chloride Methanol Coomassie blue NaCl KCl Na 2 HPO 4 KH 2 PO 4 PBS (see Recipes) Gel destaining solution (see Recipes) 50 mM 12 C-IPA/DMSO solution (see Recipes) 50 mM 13 C-IPA/DMSO solution (see Recipes) 5 mM 13 C-IPA/25 mM ammonium bicarbonate (see Recipes) 25 mM ammonium bicarbonate buffer (see Recipes) 25 mM ammonium bicarbondate/50% acetonitrile buffer (see Recipes) 25 mM ammonium bicarbonate/10 mM calcium chloride buffer (see Recipes) 1 M calcium chloride solution (see Recipes) 40 mM DTT/25 mM ammonium bicarbonate (see Recipes) 1 mM HCl solution (see Recipes) 50 mM acetic acid (see Recipes) 5% formic acid (see Recipes) 5% formic acid/50% acetonitrile buffer (see Recipes) 1% trifluoroacetic acid solution (see Recipes) Wetting solution (see Recipes) Equilibration solution (see Recipes) 0.1% formic acid (see Recipes) HPLC Buffer A (see Recipes) HPLC Buffer B (see Recipes)

Techniques: Sequencing