Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 1. BLU-VIPR allows for optogenetic CRISPR using light-induced expression of gRNA. ( A ) The RGR design consists of an HH ribozyme followed by the gRNA and an HDV ribozyme. After precise self-cleavage of the ribozymes, a functional gRNA is released. ( B ) Design of construct for VPR-EL222-dependent activation of C120 promoter transcription, allowing for simultaneous expression of mCherry and gRNA after exposure to blue light (470 nm). ( C ) Comparison of mCherry reporter expression in HEK293T cells after transfection with VPR-EL222 or VP16-EL222 constructs f ollo w ed b y e xposure to blue light f or 24 h. T he e xperiment w as perf ormed three times and one representativ e image is sho wn. Scale bars are 100 μm. ( D ) Out-of-frame Cas9 reporter where the expression of tdTomato is restored after Cas9-mediated indels. ( E ) HEK293T cells were transfected with BLU-VIPR (without mCherry) containing a gRNA targeting the out-of-frame sequence in tdTomato and Cas9. After 48 h of light exposure, the cells were cultured for an additional 72 h before detection of tdTomato demonstrating successful optogenetic induction of indels by Cas9. Scale bars are 55 μm. T he e xperiment w as perf ormed three times and one representativ e image is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: CRISPR, Expressing, Functional Assay, Construct, Activation Assay, Comparison, Transfection, Sequencing, Cell Culture

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 4. Optogenetic gene editing of primary mouse Cas9 + T lymphocytes in vitro . ( A ) Design of the retroviral MSCV-BLU-VIPR constructs for blue-light induced expression of gRNA after transduction of primary mouse T lymphocytes. ( B ) Cas9 + splenic mouse T lymphocytes (Thy1.2 + ) were transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA, f ollo w ed b y e xposure to blue light and analy sis b y flo w cytometry. ( C ) After 48 h of light exposure, followed by a 72-h dark period, the T lymphocytes were stained for Thy1.2, gated for singlets and viability, and then analyzed for T h y1.2 e xpression. R epresentativ e result from tw o e xperiments is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vitro, Retroviral, Construct, Expressing, Transduction, Cytometry, Staining

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 5. BLU-VIPR allows for optogenetic gene editing of Cas9 + T lymphocytes in vivo . ( A ) Optogenetic setup for illumination of lymph nodes with blue light (470 nm). ( B ) Illumination of a single iLN. ( C ) Cas9 + splenic mouse T lymphocytes (CD45.1 + T h y1.2 + ) w ere transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA. T he transduced cells w ere adoptiv ely transferred to TCR β−/ −CD45.2 + mice. After reconstitution of the T cell lymphocyte pool, we illuminated an iLN with blue light to induce gene editing of Thy1.2. ( D ) Gating strategy to determine levels of Thy1.2 by flow cytometry. ( E ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from illuminated iLNs were determined by flow cytometry. ( F ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from nonilluminated mice w ere determined b y flo w cytometry. ( G ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) in blood bef ore and after illumination of an iLN w ere determined b y flo w cytometry.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vivo, Transduction, Flow Cytometry, Cytometry

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: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

Techniques: Quantitative Proteomics, Gene Expression, Expressing, SDS Page, Western Blot, Control

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: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

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

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: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

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: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Control, 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: Genetic ablation of LASP1 impairs Matrigel invasion ability and alters the EMT program in TNBC cells. ( A ) LASP1 was genetically edited out by employing the CRISPR-Cas9 approach in MDA-Bone-Un cells. Left panel: The CC and LASP1-KO MDA-Bone-Un cells were plated in DMEM containing 10% serum. The light micrographs depict the morphology of CC and LASP1-KO cells. Right panel: the knockout of LASP1 was confirmed by Western blotting of total cell lysates of MDA-Bone-Un cells. Moreover, β-tubulin served as the loading control. Scale bar: 50 µM. ( B ) The CC and LASP1-KO MDA-Bone-Un cells (1 × 10 5 cells) were seeded onto the upper chamber and allowed to invade through the Matrigel towards the lower chamber containing 25 nM CXCL12 as the chemoattractant overnight. The Matrigel, along with non-invaded cells in the upper chamber, were cleared off with a rubber policeman. The invaded cells that adhered to the bottom surface were fixed and stained with 0.1% crystal violet. The representative images for each condition were depicted (left panel). The cells in five randomly selected fields were counted and expressed as “Mean ± SD”, and the fold invasion is calculated and plotted (right panel); n = 3. Scale bar: 50 µM. ( C,D ) Altered epithelial and mesenchymal marker profile upon knockout of LASP1. Moreover, 25 µg of total cell lysate from CC and LASP1-KO in MDA-Bone-Un and HEK-293 cells were separated by 10% SDS-PAGE and the indicated epithelial and mesenchymal markers were analyzed by immunoblotting. β-tubulin served as the loading control. ( E ) Following LASP1 knockout in MDA-Bone-Un cells, ALDH activity was measured by FACS analysis. The given values represent mean ± SEM in 3 biological replicates ( n = 3). ( F ) Altered profile of Snail1 and proteins involved in its stability upon knockout of LASP1. 25 µg of nuclear lysates from CC and LASP1-KO in MDA-Bone-Un cells were separated by 10% SDS-PAGE and analyzed by immunoblotting. Lamin A/C served as the loading control. ( G ) Mislocalization of Snail1 upon genetic ablation of LASP1 in MDA-Bone-Un cells. Cells seeded onto coverslips that were coated with collagen IV were serum-starved for 1 h and fixed. The subcellular localization of Snail1 (pseudo-colored green) was examined by immunofluorescent staining. DRAQ5 and Rhodamine-Phalloidin were employed to mark the nuclei and F-actin respectively. Confocal microscopic images were acquired and the distribution of the Snail1 was quantified using Image J software. Scale bar: 50 µM. Representative confocal micrographs of the cells that were subjected to quantification is depicted (left panel). The middle panel profiles the peak distribution of Snail1 in the representative images in the cytoplasm and nuclei of CC and LASP1-KO cells. The right panel displays the plots with fold change in the distribution of the Snail1 for CC and LASP1-KO cells. CC—CRISPR control, KO—LASP1 knock out. * p < 0.05.

Article Snippet: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

Techniques: CRISPR, Knock-Out, Western Blot, Control, Staining, Marker, SDS Page, Activity Assay, Software

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: In order to generate knockout (KO) of LASP1 in MDA-Bone-Un and HEK-293 cells, LASP1 CRISPR/Cas9 knockout (LASP1-KO) plasmids (a set of 3 plasmids) were obtained (Santa Cruz, Dallas, TX, USA; Cat. No. sc-404630).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Ubiquitin Proteomics, Gene Expression, Translocation Assay, Inhibition