Journal: British journal of cancer

Article Title: Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

doi: 10.1038/sj.bjc.6605850

Figure Lengend Snippet: Figure 7 Knockdown of WASF3 expression suppresses MMP9 expression levels and inhibits MMP9 release from prostate cancer cell lines. (A) RT–PCR analysis of PC3 and DU145 cells showing knockdown of WASF3 also show down-regulation of MMP9, but not MMP2 expression levels. Cont ¼ parental cells. (B) Secretion of the pro- and active form of MMP9 from PC3 and DU145 cells were measured in the supernatants of actively growing cells using the Fluorokine MAP assay procedure. Cells from each clone showing knockdown of WASF3 also showed reduced MMP9 levels compared with parental (control) and non-knockdown cells that did not.

Article Snippet: After 24 h, the media were collected in tubes and centrifuged for 10 min at 10 000 g. The pro- and active MMP-9 levels released into the media were measured using a Fluorokine MAP human MMP9 kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 1 Loss of Pals1 in a colorectal cancer cell line HCT116 results in TJ defects, enhanced migration and invasion. A Immunostaining of confluent HCT116 and HCT116ΔPals1 cells with indicated antibodies, B Activation of Rac1 in wild type and Pals1-deficient HCT116 was quantified using G-LISA assay. C Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in HCT116 and HCT116ΔPals1 cells. Results are representative of 4 experiments. D Quantification of Rac1 biosensor FRET signals at the cell body and the cell cortex in HCT116 and HCT116ΔPals1 cells. Scale bars are 20 µm in A, C.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Activation Assay, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 2 Deletion of Pals1 in Caco-2 cells does not result in enhanced migration and invasion or upregulation of active Arf6 or Rac1. A Immunostaining of confluent Caco-2 and Caco-2ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of Caco-2 and Caco-2ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasions assays of Caco-2 and Caco-2ΔPals1 cells (N = 5). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 3). F Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in Caco-2 and Caco-2ΔPals1 cells. Results are representative of 4 experiments. Scale bars are 20 µm in A and F, 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Western Blot, Staining, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 3 Pals1-deficient DLD1 do not exhibit increased Arf6/Rac1 activity or enhanced cell migration/invasion. A Immunostaining of confluent DLD1 and DLD1ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of DLD1 and DLD1ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasion assays of DLD1 and DLD1ΔPals1 cells (N = 3). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 8). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 3). Scale bars are 20 µm in A and 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activity Assay, Migration, Immunostaining, Western Blot, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 4 Knockout of Pals1 in mesenchymal-like RKO cells does not affect cell motility. A Western blot analysis of the expression of E-Cadherin in different colorectal cancer cell lines. B Representative images from wound healing assays of RKO and RKOΔPals1 cells and the corresponding quantification (N = 6). C Representative images and quantification of transwell matrigel invasion assays of RKO and RKOΔPals1 cells (N = 4). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of RKO and RKOΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of RKO and RKOΔPals1 cells (N = 3). Scale bars are 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Knock-Out, Western Blot, Expressing, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 6 SW48ΔPals1 cells display enhanced Arf6/Rac1 activation and increased cell migration, which can be rescued by SMAP1 transfection. A Immunostaining of confluent SW48 and SW48ΔPals1 cells with the indicated antibodies. B Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in SW48 and SW48ΔPals1 cells. Results are representative of 3 experiments. C Western blot of cell lines with and without SMAP1 overexpression. Empty vector was used as negative control. D, E Quantification of cell migration (scratch assay, D) and invasions assay (E) of the indicated cell lines. F Rac1 activation of the indicated cell lines quantified by G-LISA. G Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of the indicated cell lines (N = 3). H Survival probability of colorectal cancer patients with only low Pals1 expression, only low SMAP1 expression or low Pals1 and low SMAP1 expression. Scale bars are 20 µm in A and B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activation Assay, Migration, Transfection, Immunostaining, Western Blot, Over Expression, Plasmid Preparation, Negative Control, Wound Healing Assay, Staining, Expressing