Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: CXCR4 and Snail1 are expressed differentially in human breast cancer cell lines. ( A ) Top panel—Kaplan–Meier analysis of the relapse-free survival rate of basal-like, triple-negative breast cancer patients with grade 3 tumors, with a differential expression of LASP1 and SNAI1 ( n = 112). Bottom panel—Beeswarm plot of differential gene expression profile of LASP1 and SNAI1 —red symbols—high expression; black symbols—low expression ( B ). Proteins in the total cell lysates were separated by 10% SDS-PAGE, and subjected to Western blot analysis for differential expression of CXCR4 by probing with anti-CXCR4 antibody in luminal and basal-like breast cancer cell lines. Moreover, β-tubulin served as the loading control ( C ) Proteins in the total cell lysates were separated by 10% SDS-PAGE and subjected to Western blot analysis for differential expression of proteins involved in Snail1 stabilization such as LASP1, LSD1, Snail1, and A20 in luminal and basal-like breast cancer cell lines. Furthermore, β-tubulin served as the loading control.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Expressing, SDS Page, Western Blot

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: Nuclear Snail1 is stabilized upon activation of CXCR4 by CXCL12. ( A ) MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 0–30 min. The harvested ( A ) total protein lysates or ( B ) nuclear lysates were separated by 10% SDS-PAGE and subjected to Western blot analysis. The protein levels of Snail1 and proteins implicated in the Snail1 stabilization, such as AKT, GSK-3β, A20, LASP1, and LSD1 were analyzed and expressed as mean ± SD for 3 independent experiments ( n = 3). Moreover, β-tubulin and Lamin A/C levels were employed as loading controls for total cell and nuclear lysate respectively. ( C ) MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/ CXCR4-ΔCTD total protein lysates were subject to Western blotting. The protein levels of AKT, GSK-3β, LSD1, A20, LASP1, Snail1, and other EMT makers were measured and normalized to the levels of the β-tubulin as the loading control. The values were expressed as mean ± SD for 3 independent experiments ( n = 3).

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Activation Assay, SDS Page, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: Domains of LASP1 differentially associate with Snail1 and its stabilizing proteins. ( A ) Top panel–Affinity purified full length LASP1 and its domains fused to GST were separated by 10% SDS-PAGE. Proteins were visualized by staining the gel with Imperial Blue Protein Stain; Bottom panel—A schematic depiction of the constructs of recombinant full length of LASP1 and its various domains that were bacterially expressed ( B ) Association of LASP1 and its domains with Snail1 upon stimulation of stably expressed CXCR4 by CXCL12. Notably, 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 200 µg of nuclear lysate derived from 293-CXCR4 cells that were transiently expressing Flag-Snail1-WT and stimulated with 20 nM CXCL12. The association of Flag-Snail1 with full length LASP1 and its domains were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3 ( C ) and ( D ) Association of LASP1 and its domains with proteins that contribute to the stabilization of the Snail1. Nuclear lysates were prepared from MDA-Bone-Un cells that were serum-starved and stimulated with 20 nM CXCL12. 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The association of endogenous A20, LSD1, AKT and GSK-3β with full length LASP1 and its domains were analyzed by 10% SDS-PAGE followed by immunoblotting; n = 3.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Affinity Purification, SDS Page, Staining, Construct, Recombinant, Stable Transfection, Incubation, Derivative Assay, Expressing, Western Blot

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p < 0.05.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: A working model illustrating the role of CXCR4-LASP1 axis in promoting the stabilization of nuclear Snail1. Left panel: A proposed model illustrating the cellular situation when CXCL12 is absent—In the absence of any activation of CXCR4 by CXCL12, the constitutively phosphorylated LASP1 at S146 (pS146-LASP1) is bound to inactive, unliganded CXCR4 at the plasma membrane. Phosphorylation of Snail1 by GSK-3β on specific residues results in cytosolic localization of Snail1. Cytoplasmic Snail1 undergoes additional phosphorylation by GSK-3β, which marks Snail1 for ubiquitination and subsequent proteasomal degradation. Gene expression of E-cadherin is not repressed by Snail1, though repression by other transcription factors such as ZEB1 is not ruled out. This happens in epithelial cancer cells. Right panel: Activation of CXCR4 by CXCL12 leads to the phosphorylation of LASP1 at Y171 and translocation of LASP1. Additionally, active CXCR4 induces phosphorylation/activation of Akt (pS473-AKT) through the PI3K pathway, which traverses to the nucleus and initiates the phosphorylation/inhibition of nuclear GSK-3β (pS9-GSK-3β). Concurrently, the CXCR4 signaling increases the nuclear levels of A20 and LSD1. Each of these proteins induces the stabilization of Snail1 in a variety of ways. A20 mediates monoubiquitination of Snail1 thus reducing its affinity for GSK-3β. Nuclear shuttled LASP1 and elevated LSD1 level may physically shield Snail1 and prevent access of GSK-3β to Snail1. The association of pLASP1 with Snail1, pAKT, A20 and LSD1 in the form of several complexes of differing compositions will protect and stabilize Snail1. The stabilized Snail1 represses the promoter region of E-cadherin. This happens in mesenchymal cancer cells. Snail1: Snail family transcriptional repressor; LASP1: LIM and SH3 protein 1; Akt: Protein kinase B; GSK-3β: Glycogen synthase kinase-3β; LSD1: lysine-specific histone demethylase 1; A20: TNF-α induced protein 3; E-cad: E-cadherin; Ub: Ubiquitination.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Activation Assay, Expressing, Translocation Assay, Inhibition

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: CXCR4 and Snail1 are expressed differentially in human breast cancer cell lines. ( A ) Top panel—Kaplan–Meier analysis of the relapse-free survival rate of basal-like, triple-negative breast cancer patients with grade 3 tumors, with a differential expression of LASP1 and SNAI1 ( n = 112). Bottom panel—Beeswarm plot of differential gene expression profile of LASP1 and SNAI1 —red symbols—high expression; black symbols—low expression ( B ). Proteins in the total cell lysates were separated by 10% SDS-PAGE, and subjected to Western blot analysis for differential expression of CXCR4 by probing with anti-CXCR4 antibody in luminal and basal-like breast cancer cell lines. Moreover, β-tubulin served as the loading control ( C ) Proteins in the total cell lysates were separated by 10% SDS-PAGE and subjected to Western blot analysis for differential expression of proteins involved in Snail1 stabilization such as LASP1, LSD1, Snail1, and A20 in luminal and basal-like breast cancer cell lines. Furthermore, β-tubulin served as the loading control.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Expressing, SDS Page, Western Blot

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: Nuclear Snail1 is stabilized upon activation of CXCR4 by CXCL12. ( A ) MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 0–30 min. The harvested ( A ) total protein lysates or ( B ) nuclear lysates were separated by 10% SDS-PAGE and subjected to Western blot analysis. The protein levels of Snail1 and proteins implicated in the Snail1 stabilization, such as AKT, GSK-3β, A20, LASP1, and LSD1 were analyzed and expressed as mean ± SD for 3 independent experiments ( n = 3). Moreover, β-tubulin and Lamin A/C levels were employed as loading controls for total cell and nuclear lysate respectively. ( C ) MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/ CXCR4-ΔCTD total protein lysates were subject to Western blotting. The protein levels of AKT, GSK-3β, LSD1, A20, LASP1, Snail1, and other EMT makers were measured and normalized to the levels of the β-tubulin as the loading control. The values were expressed as mean ± SD for 3 independent experiments ( n = 3).

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Activation Assay, SDS Page, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: Domains of LASP1 differentially associate with Snail1 and its stabilizing proteins. ( A ) Top panel–Affinity purified full length LASP1 and its domains fused to GST were separated by 10% SDS-PAGE. Proteins were visualized by staining the gel with Imperial Blue Protein Stain; Bottom panel—A schematic depiction of the constructs of recombinant full length of LASP1 and its various domains that were bacterially expressed ( B ) Association of LASP1 and its domains with Snail1 upon stimulation of stably expressed CXCR4 by CXCL12. Notably, 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 200 µg of nuclear lysate derived from 293-CXCR4 cells that were transiently expressing Flag-Snail1-WT and stimulated with 20 nM CXCL12. The association of Flag-Snail1 with full length LASP1 and its domains were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3 ( C ) and ( D ) Association of LASP1 and its domains with proteins that contribute to the stabilization of the Snail1. Nuclear lysates were prepared from MDA-Bone-Un cells that were serum-starved and stimulated with 20 nM CXCL12. 1.5 nmol of each of the GST and LASP1 and its domains fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The association of endogenous A20, LSD1, AKT and GSK-3β with full length LASP1 and its domains were analyzed by 10% SDS-PAGE followed by immunoblotting; n = 3.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Affinity Purification, SDS Page, Staining, Construct, Recombinant, Stable Transfection, Incubation, Derivative Assay, Expressing, Western Blot

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p < 0.05.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

doi: 10.3390/cancers12092372

Figure Lengend Snippet: A working model illustrating the role of CXCR4-LASP1 axis in promoting the stabilization of nuclear Snail1. Left panel: A proposed model illustrating the cellular situation when CXCL12 is absent—In the absence of any activation of CXCR4 by CXCL12, the constitutively phosphorylated LASP1 at S146 (pS146-LASP1) is bound to inactive, unliganded CXCR4 at the plasma membrane. Phosphorylation of Snail1 by GSK-3β on specific residues results in cytosolic localization of Snail1. Cytoplasmic Snail1 undergoes additional phosphorylation by GSK-3β, which marks Snail1 for ubiquitination and subsequent proteasomal degradation. Gene expression of E-cadherin is not repressed by Snail1, though repression by other transcription factors such as ZEB1 is not ruled out. This happens in epithelial cancer cells. Right panel: Activation of CXCR4 by CXCL12 leads to the phosphorylation of LASP1 at Y171 and translocation of LASP1. Additionally, active CXCR4 induces phosphorylation/activation of Akt (pS473-AKT) through the PI3K pathway, which traverses to the nucleus and initiates the phosphorylation/inhibition of nuclear GSK-3β (pS9-GSK-3β). Concurrently, the CXCR4 signaling increases the nuclear levels of A20 and LSD1. Each of these proteins induces the stabilization of Snail1 in a variety of ways. A20 mediates monoubiquitination of Snail1 thus reducing its affinity for GSK-3β. Nuclear shuttled LASP1 and elevated LSD1 level may physically shield Snail1 and prevent access of GSK-3β to Snail1. The association of pLASP1 with Snail1, pAKT, A20 and LSD1 in the form of several complexes of differing compositions will protect and stabilize Snail1. The stabilized Snail1 represses the promoter region of E-cadherin. This happens in mesenchymal cancer cells. Snail1: Snail family transcriptional repressor; LASP1: LIM and SH3 protein 1; Akt: Protein kinase B; GSK-3β: Glycogen synthase kinase-3β; LSD1: lysine-specific histone demethylase 1; A20: TNF-α induced protein 3; E-cad: E-cadherin; Ub: Ubiquitination.

Article Snippet: After this, the membranes were washed with TBST and incubated with the following primary antibodies in 5% BSA or NFDM for 2 h at 4 °C: LASP1 8C6 clone (Biolegend, San Diego, CA, Cat. No. 909301), Snail1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3879), A20 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 4625), A20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. 166692), LSD1 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 2139), CXCR4 (BD Bioscience, San Jose, CA, USA, Cat. No. 551852), AKT (Cell signaling Technology, Danvers, MA, USA, Cat. No. 9272), p-AKT S473 (Cell signaling Technology, Danvers, MA, USA, Cat. No. 3527), GSK-3β (Cell signaling Technology, Cat. No. 12456), p- GSK-3β S9 (Cell signaling Technology, Cat. No. 5558), Vimentin (Cell signaling Technology, Cat. No. 5741), E-Cadherin (Cell signaling Technology, Cat. No. 14472), EpCAM (Cell signaling Technology, Cat. No. 2929), FBP1 (Cell signaling Technology, Cat. No. 59172), CK-14 (Thermo Fisher LL002, MA511599, Waltham, MA, USA), CYK 18 (Thermo Fisher LL002), Lamin A/C (Cell signaling Technology, Cat. No. 4777), and β-tubulin (Sigma/Aldrich, St. Louis, MO, Cat. No. T0198).

Techniques: Activation Assay, Expressing, Translocation Assay, Inhibition