crispr kit  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a Illustrative diagram and gel electrophoresis reproduction (4% agarose gel) revealing the PCR results indicating the 11-base pair (bp) deletion observed in Alms1 -/- mice compared to the control wild-type (WT) mice. b , c Body weights ( b ) and blood glucose levels ( c ) of the mice used in the study throughout their lifespan until 20 weeks of age. n = 10 mice ( Alms1 WT) and 12 mice ( Alms1 -/- ) per group (biological replicates). d Representative scheme of the temporal development of AöS. e , f Heatmap clustered by Euclidean distance ( e ) shows significant alterations in metabolites or metabolomic ratios in liver (log FC > ± 1). Volcano plots ( f ) highlight upregulated and downregulated metabolites. n = 8 mice per group (biological replicates). Full data in Supplementary Data . g , h Hepatic gene expression changes shown in heatmap clustered by Euclidean distance ( g ) and volcano plot ( h ) with log FC > ± 1. n = 7 mice per group (biological replicates). Full data in Supplementary Data . The data are reported as the mean ± SEM ( b , c ), volcano plots with individual metabolite/gene values are depicted as circles. Each dot represents one metabolite/gene, and exact P- values are shown on the figure. Source data are provided as a Source Data file. P- values were calculated with a two-way mixed-effects model and adjusted using Sidak’s correction (two-sided) ( b , c ), and metabolites were tested with two-sided t tests, with P- values adjusted for multiple testing using the Benjamini–Hochberg FDR procedure ( e – h ).

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques: Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Control, Gene Expression

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a – c Heatmaps depicting all changes in: (i) Alms1 -/- vs WT mice; (ii) DIO-mice vs normal diet (ND) mice ( a ). Correlations report r and Pearson correlation P between revealed alterations between DIO and Alms1 -/- mice ( b ). Venn diagram showing unique metabolic alterations in Alms1 -/- mice ( c ). n = 5 mice per group (biological replicates). Full data in Supplementary Data . d , e Significant alterations in hepatic metabolite concentration or metabolite ratios in the liver at 20 weeks are shown in a heatmap clustered by Euclidean distance, depicting changes with log 2 FC > ± 1 ( d ). Volcano plots ( e ) highlight upregulated metabolites in red and downregulated in blue. n = 8 mice per group (biological replicates). Full data in Supplementary Data . f , g Significant alterations in hepatic gene expression at 20 weeks are shown in a heatmap clustered by Euclidean distance, depicting changes with log 2 FC > ± 1 ( f ). Volcano plots ( g ) highlight upregulated genes in red and downregulated in blue. n = 8 mice per group (biological replicates). Full dataset available in Supplementary Data . Each dot represents one metabolite/gene and the exact P- values are shown in the figure. The data are reported as the mean ± SEM, heatmaps or volcano plots with individual mouse or item analyzed values depicted as circles. The quantity of mice ( n ) used in each experiment and exact P- values are shown on the figure. Source data are provided as a Source Data file. P- values were calculated using two-sided Pearson correlation ( b ), and metabolites were tested with two-sided t tests, with P- values adjusted for multiple testing using the Benjamini–Hochberg FDR procedure ( d , g ). Abbreviations used: DIO Diet-induced obesity. HFD High-fat diet.

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques: Concentration Assay, Gene Expression

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a – d Representative western blot (WB) images of ACBP, SQSTM1, LC3-II/LC3-I, GAPDH and Stain-free gel levels ( a ) with quantifications for ACBP ( b ), SQSTM1 ( c ), and the LC3-II/LC3-I interconversion ratio ( d ). n = 9 mice per group (biological replicates). e – g Representative WB images of ACBP, SQSTM1 and GAPDH ( e ) with respective quantifications for ACBP ( f ) and autophagy flux calculation as the ratio SQSTM1(Leu)/SQSTM1(Unt) ( g ). n = 3 mice per group. h , i ACBP levels in plasma in mice aged 6- or 20-week-old, WT or Alms1 -/- ( h ), under ad libitum feeding conditions or after 24 h period of caloric restriction (CR) ( i ). n = 5 mice (WT and Alms1 -/- ad libitum ), n = 8 mice ( Alms1 WT, CR) and). n = 10 mice ( Alms1 -/- , CR) per group. j , k Representative WB images of FASN, SQSTM1, ACBP and GAPDH ( j ) in primary hepatocytes isolated from mice WT or Alms1 -/- and respective quantifications ( k ). n = 3 mice per group (biological replicates). l , m Representative WB images of ALMS1, ACBP and GAPDH ( l ) in HepG2 cells silenced (knockdown, KD) with an unrelated control (UNR) or two specific siRNA sequences targeting ALMS1 , ± bafilomycin A1 (BafA1) for 4 h ( m ). n = 4 independent experiments. n – p PCR verification of ALMS1 knockout (KO) in HepG2 cells or their wild-type (WT) control ( n ) and representative western blot images of ACBP and GAPDH ( o ) in 2 clones for ALMS1 KO HepG2 cell line, or respective control WT, ± BafA1 for 4 h ( p ). n = 3 independent experiments. All WB quantifications were normalized using GAPDH as a loading control. The data are represented as the mean ± SEM, with individual mouse/replicate values depicted as circles, and exact P- values are shown on the figure. Source data are provided as a Source Data file. P- values were calculated using two-sided unpaired t tests ( b – d , g – h , k ). For panels with multiple comparisons, P- values were adjusted (two-sided) using the Sidak method ( f , i ) or Tukey’s test ( m , p ). Abbreviations used: Bp base pair; kDa kilodalton; MW molecular weight; r.u .: relative units; Unr unrelated; empty control vector for gene silencing as a reference.

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques: Western Blot, Staining, Clinical Proteomics, Isolation, Knockdown, Control, Knock-Out, Clone Assay, Molecular Weight, Plasmid Preparation

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a , b Body weight in wild-type (WT) and Alms1 -/- mice treated or not with anti-ACBP for 4 weeks of injections initiated when they were AöS symptomatic (therapeutic approach) ( a ) or prior to develop symptoms associated with AöS (preventive approach) ( b ). n = 5 (all groups, curative approach and Alms1 WT and Alms1 -/- , preventive approach) and n = 6 mice (treated Alms1 -/- , preventive approach) per group (biological replicates). c Glucose tolerance test (GTT), with analysis of the area under the curve (AUC) for statistical comparison. n = 5 mice per group. d , e Hepatic steatosis levels ( d ), and adipocyte size ( e ) measured after preventive anti-ACBP mAb treatment. n = 5 ( Alms1 WT and Alms1 -/- ) and n = 6 mice (treated Alms1 -/- ) per group. f , g Representative western blot images of FASN, SQSTM1 and GAPDH ( f ) after anti-ACBP mAb preventive treatment, with quantifications for FASN and SQSTM1 ( g ). n = 5 ( Alms1 WT and Alms1 -/- ) and n = 6 mice (treated Alms1 -/- ) per group. h , i Representative western blot images of FASN, SQSTM1, ACBP, and GAPDH in primary hepatocytes isolated from mice with or without the Alms1 mutation, treated for 4 h with anti-ACBP mAb ( h ) and respective FASN level quantification ( i ). All western blots quantifications are normalized using GAPDH as a loading control. n = 3 mice per group. j , k Incorporation of 13 C into hepatic palmitic acid from 13 C-labeled glucose ( j ) and its correlation with the weight of mice ( k ). n = 5 ( Alms1 WT and treated Alms1 -/- ) and n = 4 mice ( Alms1 -/- ) per group (biological replicates). Each dot represents one mouse, and exact P- values are shown on the figure. Source data are provided as a Source Data file. The results are represented as the means ± SEM, with individual mouse values for each experiment depicted as circles. P -values were calculated using one-way ANOVA with Tukey’s multiple comparisons test (two-sided) ( a – j ). Correlations were assessed with Pearson’s r (two-sided) (k). Abbreviations used: FASN fatty acid synthase, g grams; GAPDH Glyceraldehyde-3-phosphate dehydrogenase; kDa Kilodalton; r.u . relative units; SQSTM1 sequestosome 1.

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques: Comparison, Western Blot, Isolation, Mutagenesis, Control, Labeling

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a – d Correlations report r and Pearson correlation P of hepatic significant changes in metabolomics ( a ), transcriptomic ( b ), proteomic ( c ) and combined analysis ( d ) between Alms1 -/- vs WT mice ( X -axis) or anti-ACBP-treated Alms1 -/- vs untreated Alms1 -/- mice ( Y -axis). e – g Venn diagram showing overlap in liver metabolomic ( e ), transcriptomic ( f ), proteomic ( g ) significant changes between Alms1 -/- vs WT mice and anti-ACBP-treated Alms1 -/- vs untreated Alms1 -/- mice. Full datasets available in Supplementary Data , , and . n = 5 ( Alms1 WT and Alms1 -/- ) and n = 6 mice (treated Alms1 -/- ) per group (biological replicates). Each dot represents one metabolite, gene or protein, and exact P- values are shown on the figure. Source data are provided as a Source Data file. The results are reported as correlation plots and Venn diagrams with individual values depicted as circles. Correlations were assessed with Pearson’s r (two-sided) ( a – d ).

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques:

Journal: Nature Communications

Article Title: Identification of ACBP as a potential target in ciliopathic obesity through multi-omics network analysis

doi: 10.1038/s41467-025-66235-4

Figure Lengend Snippet: a , b Schematic representation of the experimental groups (a) and heatmap showing the relative abundance of genera with at least one significant change across groups ( b ). c , d Venn diagrams illustrating the microbiota changes associated with Alms1 -/- mutation ( c ) or general obesity ( d ). e , f Relative abundance observed in the fecal samples of the analyzed mice. Taxa modulated between WT and Alms1 -/- mice are shown based on the LDA analysis ( e ), along with their relative abundance following anti-ACBP mAb treatment ( f ). n = 5 (3-week-old cohorts) and n = 6 mice (10-week-old cohorts) per group (biological replicates). Full dataset available in Supplementary Data . The results are represented as the means ± SEM, with individual mouse values for each experiment depicted as circles. and exact P- values are shown on the figure. Source data are provided as a Source Data file. P- values were determined using two-sided Kruskal-Wallis rank ( c , d ), LDA analysis ( e ), or one-way ANOVA with Tukey’s multiple comparisons test (two-sided) ( f ). Abbreviations used: DIO Diet-induced obesity. HFD High-fat diet. LDA linear discriminant analysis.

Article Snippet: The CRISPR/Cas9 ALMS1 -encoding plasmids ( KN414632 , OriGene) were also applied by lipofectamine transfection according to the manufacturer’s instructions, and cells were analyzed after puromycin (P8833, Sigma) selection.

Techniques: Mutagenesis

Journal: Cell Death Discovery

Article Title: Salmonella typhimurium co-expressing cytolysin A and hyaluronidase suppresses tumor growth and metastasis

doi: 10.1038/s41420-025-02897-9

Figure Lengend Snippet: A Quantification of HA levels in cell culture medium. Cells (2 × 10⁵) were seeded in 12-well plates. After 24 h, the culture medium was collected and analyzed using an HA quantification kit. B Expression of CD44, the mesenchymal marker vimentin, and the epithelial marker E-cadherin by different cancer cell types. Cell lysates (20 µg) were subjected to western blot analysis with antibodies against CD44, vimentin, and E-cadherin. β-actin was used as a loading control. C HysA activity of CNC018pCH. CNC018pCH was streaked onto an HA agar plate with or without 300 ng/mL Doxy and incubated overnight at 37 °C. HA acid-degraded clear zones were observed after acetic acid treatment. D Quantification of HysA activity in CNC018pCH supernatants. After Doxy induction (300 ng/mL for 5 h), bacterial supernatants were analyzed in an HysA activity assay. Activity was expressed as turbidity reduction units (TRU). E HA degradation in cell culture media by HysA-secreting bacteria. 2 × 10⁵ MDA-MB-231 or HCT116 cells were treated with the supernatants of engineered CNC018 strains for 24 h. 100 units/mL of purified HysA was used as a positive control. The concentration of HA was measured using an ELISA kit. F Invasion assay. Cells (3 × 10⁴/well) were seeded in 24-well Matrigel invasion chambers and treated with 100 units/mL purified HysA for 24 h. Invaded cells were stained and counted under a light microscope. G Representative images of the invasion assay in ( F ). Scale bar: 100 μm. H Phospho-kinase array analysis. MDA-MB-231 or HCT116 cells (3 × 10⁵/well) were seeded in 12-well plates and treated with 100 units of purified HysA for 24 h. Cell lysates (200 µg protein) were analyzed using a phospho-kinase array kit (R&D Systems, ARY003C). Please also see the phospho-kinase array coordinate map in Fig. . I Phospho-kinase expression in high and low metastatic cancer cells. Cell lysates (20 µg) were analyzed by Western blotting with anti-RSK antibodies. J Phospho-kinase expression in HA-downregulated MDA-MB-231 cells. The HAS2 gene was knocked out using CRISPR/Cas9. Cell lysates (20 µg) were analyzed by western blotting with anti-RSK antibodies. WT: wild-type MDA-MB-231 cells; HAS2⁻/⁻: HAS2 -knockout MDA-MB-231 cells. Data are presented as mean ± s.e.m. from three independent experiments. Statistical analyses were performed using one-way ANOVA ( A ), unpaired two-tailed t-test ( D ), two-way ANOVA ( E , F ), with Tukey’s post-hoc test where applicable.

Article Snippet: The HAS2 gene was deleted using the CRISPR-Cas9 KN2.0 non-homology-mediated method obtained from Origene Technologies Inc (USA).

Techniques: Cell Culture, Expressing, Marker, Western Blot, Control, Activity Assay, Incubation, Bacteria, Purification, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Invasion Assay, Staining, Light Microscopy, CRISPR, Knock-Out, Two Tailed Test

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: Expression of a non-canonical Wnt ligand, Wnt5a, is modulated by WNT974 in Ewing sarcoma cells. ( A ) Ewing sarcoma cell lines were treated with or without WNT974, and expression of Wnt5a was determined by quantitative RT-PCR. Error bars represent standard error of the mean of triplicate experiments, and * indicates a p value < 0.05. ( B ) Wnt5a mRNA levels were evaluated by quantitative RT-PCR in Ewing sarcoma PDX tumors growing in NSG mice treated with either DMSO (control) or WNT974. Error bars represent standard error of the mean of triplicate experiments, and p values are indicated. ( C ) Wnt5a protein expression was evaluated by immunohistochemistry in Ewing sarcoma PDX tumors from NSG mice treated with DMSO or WNT974. Panels (A and B) are from individual tumors. Magnification is indicated in each frame. ( D ) TOPFlash luciferase assay demonstrates high activity only in the presence of recombinant Wnt3a (1 μg/mL and 250 ng/mL), and little to no activity in the presence of Wnt5a (1 μg/mL, 500 ng/mL, and 250 ng/mL, respectively). ( E ) Wnt5a mRNA expression was evaluated by quantitative RT-PCR in Ewing sarcoma cell lines treated with or without WNT974, as well as increasing concentrations of exogenous recombinant Wnt5a protein (1 μg/mL, 500 ng/mL, and 250 ng/mL, respectively). A4573 (i) and CHLA10 (ii) cells show a distinct response pattern compared with the nonmetastatic CHLA9 cell line (iii). Each experiment was repeated a minimum of three times. Error bars represent standard error of the mean of triplicate experiments, and asterisks indicate the degree of statistical difference between indicated conditions * p

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: Expressing, Quantitative RT-PCR, Control, Immunohistochemistry, Luciferase, Activity Assay, Recombinant

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: Ewing sarcoma cell migration is regulated by Wnt5a. ( A ) WNT974 decreased the migration of Wnt5a-responsive Ewing sarcoma cell lines A4573, TC71, and CHLA10, but not the migration of the unresponsive cell lines CHLA9 and SK-ES-1, towards FBS in Boyden chamber assays. ( B ) Recombinant Wnt5a, but not recombinant Wnt3a, is a strong chemoattractant to A4573 cells in Boyden chamber assays. ( C ) Inclusion of rWnt5a in the upper chamber inhibited migration of Ewing sarcoma cells toward FBS (group A), whereas including rWnt5a in the lower chamber, along with FBS, augmented migration (group B). This effect is abolished by the inclusion of WNT974 in the upper chambers (group C). In each experiment, error bars indicate standard error of the mean, and asterisks indicate the degree of statistical significance. Each experiment was repeated a minimum of three times. * p

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: Migration, Recombinant

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: ( A ) Western blot for pFAK (Tyr 397) and pSrc (Tyr 416) in seven Ewing sarcoma cell lines treated with vehicle control (DMSO) versus WNT974, with a decrease in both pFAK and pSrc in the majority of cell lines treated with WNT974. ( B ) Immunofluorescence example for pFAK (Tyr 397) in A4573 cells. Note that pFAK (green) is found in the protrusions of the DMSO-treated A4573 cells but is found in the centralized clumps of actin (pink) in the WNT974-treated cells. With WNT974 treatment, there is a statistically significant decrease in pFAK puncta and the amount of pFAK throughout the cells. Green-fluorescent insert demonstrates pFAK with dots representative of Volocity analysis. ( C ) Boyden chamber assay using two different doses of FAK inhibitor GSK2256098 in A4573 and TC71 cells. Note that migration is significantly impaired in both cell lines upon treatment with the FAK inhibitor, even at nanomolar doses. ( D ) Treatment with Src inhibitor, saracatinib (1 μM), also results in a statistically significant decrease in cell migration, also suggesting that Src is important for Ewing sarcoma cell migration. In addition to catalytic proteins, focal adhesions contain adaptor proteins that mediate interactions between the cytoskeleton and the extracellular matrix . ALCAM is an adaptor protein that contains immunoglobulin-rich domains with the amino-terminal V-type immunoglobulin domain required for cell-to-cell adhesive interactions. ALCAM can form homotypic or heterotypic interactions, and its expression is highest at areas of cell-to-cell contact where it can interact with other cell adhesion molecules [ , ]. ALCAM is expressed in over 70% of pediatric sarcomas, so we started by assessing changes in protein expression with WNT974 treatment . ALCAM protein levels were unchanged with WNT974 treatment, so we next evaluated ALCAM distribution in the cell (Supplementary E). As shown in A, the vehicle control-treated cells maintain a small number, typically 2–3, of well-formed long protrusions in which ALCAM can be found throughout, in addition to its presence in the perinuclear region of the cytoplasm. In contrast, in cells treated with WNT974, there is bright centralized clumping of ALCAM and a notable loss of long protrusions in most of the cells. The highest concentration of ALCAM in these cells appears to overlap the nucleus, in contrast to the perinuclear cytoplasmic distribution in the vehicle control-treated cells. In addition, many of the WNT974-treated cells demonstrate a more circumferential ruffled appearance to the plasma membrane. In addition, there is a decrease in the number of long protrusions from the cells with WNT974 treatment ( A). ALCAM is a member of a family of cell adhesion molecules. Another family member that has been implicated in sarcoma biology is MCAM [ , , ]. The involvement of ALCAM in Wnt5a-mediated cytoskeletal rearrangements is specific, because no such changes were appreciated with MCAM immunofluorescence ( A). * p

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: Western Blot, Control, Immunofluorescence, Boyden Chamber Assay, Migration, Adhesive, Expressing, Concentration Assay, Clinical Proteomics, Membrane

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: ( A ) Immunofluorescence of ALCAM (green) and vinculin (red) in A4573 cells and TC71 cells. Note the co-localization of both proteins and the centralized clumping with WNT974 treatment in both cell lines. When given recombinant Wnt5a, ALCAM and vinculin both appear less bright versus if the cells are treated with WNT974 and then given that same dose of recombinant Wnt5a. This suggests that the cells respond to the amount of Wnt5a in the environment which affects ALCAM and vinculin. Additional panel demonstrating similar findings in TC71 cells. MCAM immunofluorescence does not demonstrate a redistribution pattern upon WNT974 treatment, in contrast to ALCAM. Quantification of protrusions per cell demonstrating a decrease in protrusions upon WNT974 treatment. ( B ) Phalloidin staining of filamentous actin is statistically significantly decreased in A4573 cells treated with WNT974 compared to vehicle control. ( C ) Immunoprecipitation of vinculin and ALCAM in A4573 cells demonstrating a significant increase in association between the two proteins upon WNT974 treatment. Notably, there is a complete dissociation between the proteins upon FAK inhibition treatment. Both changes are different from vehicle control-treated cells, suggesting that changes to how much these two proteins are in contact affects cell migration. ( D ) ( i ) Using palmostatin B, an inhibitor of acyl-protein thioesterase 1 which depalmitoylates cell surface receptors to allow for lateral migration through the membrane, A4573 cells demonstrate a centralized clumping of ALCAM. ( ii ) Additionally, WNT974 and palmostatin B only inhibit migration in Boyden chamber assays in CLHA10, the metastatic-derived cell line, but not in CHLA9, the localized-derived cell line. Only CHLA10 responds to the lowest amount of recombinant Wnt5a as a chemoattractant by significantly increased cell migration to approximately 400% of vehicle migration, consistent with the hypothesis that Wnt5a may be more active in the metastatic process. Each experiment was repeated a minimum of three times. Error bars represent standard error of the mean of triplicate experiments, and asterisks indicate the degree of statistical difference between indicated conditions ** p

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: Immunofluorescence, Recombinant, Staining, Control, Immunoprecipitation, Inhibition, Migration, Membrane, Derivative Assay

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: CRISPR-Cas9 gene editing confirms the critical role of Wnt5a in Ewing sarcoma migration. ( A ) Western blotting confirms that the Wnt5a CRISPR-Cas9 clones A9.5 and F10.6 lack Wnt5a protein expression and show a decrease in both vinculin and ALCAM expression as well as a shift in ALCAM banding patterns compared with the parental cells A4573. Beta-2-microglobulin (B2M) is a loading control. ( B ) Photomicrograph of Wnt5a CRISPR-Cas9 clone A9.5 which grows as clumps of cells with bright GFP+ signal (100 μm). ( C ) In Boyden chamber assays, the Wnt5a CRISPR-Cas9 clones migrate statistically significantly less than the scramble control. ( D ) In Boyden chamber assays using FBS as a chemoattractant, the Wnt5a CRISPR-Cas9 clones demonstrate impaired migration in comparison to scramble control. In panels C and D, error bars represent standard error of the mean, and asterisks reflect the degree of statistical significance. Experiments were repeated three times. ( E ) PNGase assay of ALCAM in parental A4573 cells, scramble control, and the two Wnt5a knock-out clones demonstrate a different banding pattern in the Wnt5a knock-out clones when treated with PNGase (denoted as +) compared with parental cells or scramble control. ( F ) Western blotting demonstrates a lack of phospho-FAK in the Wnt5a knock-out clones. ( G ) Immunofluorescence analysis of parental A4573 cells, scramble control cells, and the two Wnt5a knock-out clones shows that the parental cells have an average of 2–3 large protrusions and co-localization of ALCAM (green) and vinculin (red) upon treatment with vehicle control (DMSO) whereas upon WNT974 treatment, the cells appear to have decreased vinculin as well as centralized perinuclear clumping of ALCAM. The same pattern is also seen in the scramble control. In contrast, both the morphology of the Wnt5a knock-out clones A9.5 and F10.6, as well as the distribution of ALCAM and vinculin, is not affected by WNT974. The Wnt5a knock-out clones, A9.5 and F10.6, have a statistically significantly increased colocalization between ALCAM and vinculin when treated with WNT974 (quantified by MFI in the fused channel), a contrast to the decreased co-localization seen in scramble control cells treated with WNT974. To the right is quantification of co-localization. Error bars represent standard deviation. ( H ) Staining of cells with fluorescently tagged phalloidin shows significantly less filamentous actin in Wnt5a knock-out clones compared with parental cells and scramble control (60X). Treatment of parental cells and scramble control with WNT974 decreases phalloidin staining, an effect not seen in the Wnt5a knock-out clones. To the right is quantification of the phalloidin staining. Error bars represent standard deviation, and asterisks indicate the degree of statistical significance. Each experiment was repeated a minimum of three times. * p

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: CRISPR, Migration, Western Blot, Clone Assay, Expressing, Control, Comparison, Knock-Out, Immunofluorescence, Standard Deviation, Staining

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: Working model of the role of Wnt5a in Ewing sarcoma cells. Upon Wnt5a binding with its Fzd receptor(s) and potential co-receptor(s), FAK (Tyr 397) becomes phosphorylated and dimerizes to phosphorylate Src (Tyr 416). Phosphorylated Src then phosphorylates vinculin, allowing it to bind to F-actin bundles and cross-linking it to ALCAM. ALCAM is depalmitolyated by APT1 to allow it to move within the cell membrane, thereby deforming the membrane at the site of proximity to actin bundles.

Article Snippet: Using A4573, we generated Wnt5a Crispr-Cas9 clones using Origene (Rockville, MD, USA) Wnt5a Human Gene Knock-out kit with a GFP-puromycin functional cassette ( KN209206 ).

Techniques: Binding Assay, Membrane