Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Ang1 induces ARP2/3 expression in colorectal cancer cells in vitro. ( a – c ) Western blotting of ARP2/3 expression in colorectal cancer (COLO320dm, SW620 and MC38) cells in the presence or absence of Ang1 (top panel). The intensity of ARP2/3 bands ( n = 3) were quantified and normalized against GAPDH using ImageJ and represented as a fold change (bottom panel).

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Expressing, In Vitro, Western Blot

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Ang1 presence is essential for ARP2/3 expression in the cancer cells in vivo. ( a ) RepreScheme 1 and ARP2/3 staining of tumour sections generated by intrasplenic injection of mouse colorectal (MC38) cancer cells into wild type (Ang1 WT) and Ang1 knockout (Ang1 KO) mice (left panel). D: Desmoplastic ring, L: Liver tissue, T: Tumour. ( b ) Represents the Correlation between Ang1 expression in the liver tissue and ARP2/3 expression in the tumour cells using Pearson correlation analysis.

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Expressing, In Vivo, Staining, Generated, Injection, Knock-Out

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: ARP2/3 mediates Ang1-driven cancer cell motility. ( a , b ) Western blotting of ARP2/3 expression in SW620 or COLO320dm cancer cells expressing shRNA-scrambled or shRNA-ARPC3. The right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). ( c , d ) Representative scratch assay in SW620 or COLO320dm cells expressing shRNA-scrambled or shRNA-ARPC3 upon exposure to Ang1. The right panels show the corresponding wound healing ratio shown in fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Western Blot, Expressing, shRNA, Wound Healing Assay

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Tie2 mediates Ang1-dependent ARP2/3 expression in the cancer cells. ( a ) Representative images of coimmunostaining for showing ARP2/3 (green) and Tie2 (red) on FFPE tissue sections of CRCLM resected from chemonaïve patients. The bottom panel represents the cancer cells at the invading front that are in direct contact with hepatocytes. ( b ) Immunoblotting of ARP2/3 expression in MC38 colorectal cancer cells expressing shRNA-Scrambled or shRNA-Tie2. The right panel shows the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). ( c , d ) Left panels show the effect of Tie2 inhibitor (BAY-826) on ARP2/3 expression in MC38 and SW620 cancer cells upon Ang1 exposure, respectively. Right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Expressing, Western Blot, shRNA

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: PI3K/AKT pathway contributes to Ang1-dependent ARP2/3 expression. ( a ) Protein interaction networks generated using STRING database version 11 showing the interaction between Ang1 (Angpt1), TIE2 (Tek), PI3Ks (PI3K), Akt (AKT1) and ARP2/3 subunits. ( b , c ) Western blotting of ARP2/3 expression in exposed MC38 or SW620 cancer cells to recombinant Ang1 in the presence or absence of PI3K/AKT inhibitor (LY294002). Right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Expressing, Generated, Western Blot, Recombinant

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: The molecular mechanism of Ang1 function in vessel co-opting CRCLM lesions. Schematic representation of key findings in the current study. The hepatocytes of vessel co-opting lesions express high levels of Ang1. The secreted Ang1 by hepatocytes interacts with the cancer cells through Tie2, which activates PI3K/AKT followed by ARP2/3 expression, respectively. Upregulation in ARP2/3 expression increases cancer cell motility and allows them to infiltrate the liver tissue to obtain blood supply by hijacking the pre-existing vessels (vessel co-option).

Article Snippet: We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582).

Techniques: Expressing

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 3. mRNA and protein expression of novel cytoskeletal molecules in PAN rats. In relation to GAPDH, the mRNA and protein expression of Tagln (transgelin) (A, A′), Birc5 (survivin) (B, B′), Actr2 (arp2) (C, C′), Krt2–7 (cytokeratin7) (D, D′) and Vcl (vinculin) (E, E′) increased significantly at Day 10, then decreased at Day 15 in PAN rats compared to controls (Con). Data are presented as mean ± SD. N = 6. ∗P < 0.05 versus Con; ∗∗P < 0.01 versus Con.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Expressing

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 4. Immunofluorescence staining of novel cytoskeletal molecules in PAN rats. Transgelin (A), survivin (D), arp2 (G) and cytokeratin7 (J) were labelled green and showed a weak staining in normal rat glomeruli (Control). Double-labelling assays showed that transgelin (C), survivin (F), arp2 (I) and cytokeratin7 (L) colocalized with podocyte marker synaptopodin, labelled red (Synap: B, H, K), displaying a linear pattern along the glomerular capillary loops. Vinculin (M), labelled red, showed colocalization with the podocyte molecule α-actinin-4, labelled green (N), displaying a dotted, linear pattern in glomeruli (O). Bar = 20 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Immunofluorescence, Staining, Control, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 5. Immunofluorescence staining of novel cytoskeletal molecules in human kidneys. Transgelin (A1, a1, a4, a7), arp2 (C1, c1, c4, c7), and cytokeratin7 (D1, d1, d4, d7) are green. Vinculin is red (E1, e1, e4, e7). Their staining was intense in the glomeruli of patients with MCNS (a1, c1, d1, e1), FSGS (a4, d4, e4) and MN (a7, c7, d7, e7) as compared to normal glomeruli (Control: A1, C1, D1, E1), except for arp2 in FSGS glomeruli (c4). Transgelin, arp2, and cytokeratin7 colocalized with the podocyte marker synaptopodin, labelled red, in glomeruli of MCNS (a3, c3, d3), FSGS (a6, d6) and MN (a9, c9, d9). Vinculin showed complete colocalization with the podocyte molecule podocin, labelled green in control (E3), MCNS (e3), FSGS (e6) and MN (e9). The staining of survivin was negative in glomeruli of normal controls (B1), but intensive staining was revealed in MCNS (b1), FSGS (b4) and MN (b7), and mainly distributed to the perinuclear area of glomeruli and tubules of MCNS (b3), FSGS (b6) and MN (b9). Bar = 20 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Immunofluorescence, Staining, Control, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 6. (A) Distribution of novel cytoskeletal molecules in cultured podocytes. Cell nuclei were stained blue with Hoechst. In normal podocytes (Control: a–e), the staining of transgelin, survivin, arp2 and cytokeratin7 was weak and mainly distributed in cytoplasm, whereas their fluorescence intensity increased, especially along the cell membrane, in puromycin aminonucleoside-treated podocytes (PAN: f–j). The distribution of vinculin was obviously altered, displaying a dotted- and a plaque-like pattern along cell membranes. Bar = 30 µm. (B) Protein expression of novel cytoskeletal molecules in cultured podocytes. Transgelin (k), survivin (l), arp2 (m), cytokeratin7 (n) and vinculin (o) increased in PAN-treated cells compared to controls. Data are presented as mean ± SD. N = 3. Values were normalized to GAPDH. ∗P < 0.05 versus Con.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Cell Culture, Staining, Control, Fluorescence, Membrane, Expressing

Journal: Journal of cell science

Article Title: FAK, PIP5KIgamma and gelsolin cooperatively mediate force-induced expression of alpha-smooth muscle actin.

doi: 10.1242/jcs.044008

Figure Lengend Snippet: Fig. 2. FAK activation after mechanical force is independent of RhoA. (A) Top panels: immunoblot analysis of Tyr397FAK-P (pY397FAK) in collagen-coated- bead-associated proteins after force application for the indicated time intervals. β1-integrin immunoblots show loading for each bead preparation. Bottom left panels: cells were incubated with collagen beads and force was applied for 60 minutes, the optimal time for phosphorylation of Tyr397FAK. Lia1/2 β1-integrin- blocking antibody was co-incubated with collagen beads prior to force application and was used here as a β1-integrin control to test for specificity. Fibronectin and BSA-coated beads were used as alternative controls in cells treated with force for 60 minutes. F, force. Bottom center panels: immunoblots show force-induced collagen-coated-bead association of talin and Arp2 after 60 minutes of force. NF, no force. Bottom right panels: activation of Src by force was determined by immunoblots of Tyr527Src-P (pY527-Src) and Tyr416Src (pY416-Src). Note that pre-incubation of cells with the Src inhibitor SU6656 at 1 μM does not inhibit force-induced phosphorylation of Tyr397FAK. (B) Confocal microscopy after force application shows Tyr397FAK-P around collagen beads. DIC images of the same cells show the location of beads. BSA-coated beads with force application were used as non-specific controls. Arrows show the area shown at higher magnification in the insets. Line graphs show fluorescence intensity across bound beads. (C) Membrane-anchored, dominant-negative Tyr397Phe FAK (DN FAK) was transfected into cells and protein recruitment to collagen-coated beads was analyzed in bead-associated proteins. After force application, immunoblots show increased levels of Tyr397FAK-P and β-actin in bead-associated proteins in vehicle (Veh)-treated cells but not in DN-FAK-treated cells. Ratios of β-actin to integrin were computed from densitometry measurements of immunoblots (mean ± s.e.m.). FAK knockdown by siRNA showed decreased Tyr397FAK-P in relation to the amount of FAK present. (D) Cells treated with Rho inhibitor, Y27632, were assessed for phosphorylation of Tyr397FAK. (E) Rho activation was measured in cells with or without siRNA knockdown using rhotekin-binding assay and immunoblotting for RhoA. Ratios of active Rho A to total Rho A were computed from densitometry measurements of immunoblots (mean ± s.e.m.).

Article Snippet: Antibodies to Arp2, Tyr527Src-P and Tyr416Src-P were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Western Blot, Incubation, Phospho-proteomics, Blocking Assay, Control, Confocal Microscopy, Fluorescence, Membrane, Dominant Negative Mutation, Transfection, Knockdown, Binding Assay

Journal: International Journal of Cancer

Article Title: ARP2 /3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma

doi: 10.1002/ijc.35246

Figure Lengend Snippet: ARP2/3 complex affects CAFs' migration capacity. (A) Representative images displaying ARP2 and ARP3 expression differences between normal and PDAC tissue. Scale bars: 50 μm. (B) Transwell‐based cell migration assay comparing CAFs treated with or without the Arp2/3 complex inhibitor CK666. Scale bars: 100 μm (left). (C) Schematic picture of morphological changes in CAFs induced by treatment with the Arp2/3 complex inhibitor CK666, emphasizing its impact on cell shape and motility (right). (D) Photomicrographs of CAFs cultured in 2D condition, either treated or untreated with CK666. (E) Quantitative analysis of leading edge length (left), number of spontaneous protruding lamellipodia per cell (middle), and the percentage of cells with multiple protruding lamellipodia (right) in 2D‐cultured cells. (F) Fluorescence confocal images stained with phalloidin. Scale bars: 50 μm. (G) Aggregated trajectories between human CAFs and CAFs with CK666 treatment (left). Distance and velocity between human CAFs and CAFs with CK666 treatment (right).

Article Snippet: Overnight incubation at 4°C with primary polyclonal rabbit antibodies against α‐SMA (#5694, Abcam, Cambridge, UK), ARP2 (#AP3861) and ARP3 (#AP4581), both from ECM Biosciences, (Munich, Germany).

Techniques: Migration, Expressing, Cell Migration Assay, Cell Culture, Fluorescence, Staining

Journal: International Journal of Cancer

Article Title: ARP2 /3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma

doi: 10.1002/ijc.35246

Figure Lengend Snippet: ARP2/3 complex is coupled with myofibroblast differentiation. (A), (B) This UMAP plot visually represents the presence of two distinct fibroblast populations (iCAFs and myCAFs) by using the published markers. (C) The dot plot illustrates the differential expression of the Arp2/3 complex signature in iCAFs and myCAFs, shedding light on their distinct molecular characteristics (lower left). (D) Immunofluorescence co‐staining of PDAC tumor tissue with ARP3 (green), αSMA (red) and DAPI (blue). One representative image is shown ( n = 8). Scale bars: 100 μm. (E) Correlation analysis of αSMA+ and ARP3+ cells was conducted using the “Colocalization Finder” tool in ImageJ 1.5.3. (F) This model provides insight into the signaling pathways that influence the formation of iCAFs and myCAFs in PDAC: TGFβ signaling plays a key role in myCAF formation, IL1 signaling drives iCAF development. (G) The results from qRT‐PCR analysis reveal the expression levels of myCAF markers (ACTA2, TAGLN, and MYL9) when treated with TGFβ1 (20 ng/mL) and iCAF markers (CXCL1, IL6, and CCL2) when treated with IL1α (1 ng/mL) in human PSCs, both in the presence and absence of CK666. These findings are derived from three independent experiments and are presented as mean ± SEM. *, indicates statistical significance with p <.05, as determined by a Student t ‐test.

Article Snippet: Overnight incubation at 4°C with primary polyclonal rabbit antibodies against α‐SMA (#5694, Abcam, Cambridge, UK), ARP2 (#AP3861) and ARP3 (#AP4581), both from ECM Biosciences, (Munich, Germany).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Protein-Protein interactions, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: Frontiers in endocrinology

Article Title: Molecular Actions of Thyroid Hormone on Breast Cancer Cell Migration and Invasion via Cortactin/N-WASP.

doi: 10.3389/fendo.2019.00139

Figure Lengend Snippet: FIGURE 4 | T3 signals to paxillin, cortactin, N-WASP and Arp2/3 complex. (A,B) T-47D cells were incubated in the presence of 10 nM T3 for 20 min with or without silencing of paxillin with specific siRNAs and/or inhibition of cortactin and N-WASP. Actin, phospho-paxillinY118, p-cortactinY466, p-N-WASPS484/485 and p-Arp2Y237

Article Snippet: Antibodies used were: p-FAKY397 (611807), FAK (610088) (BD Transduction Laboratories, Lexington, KY); p-FAK (Tyr397) (sc-11765-R), cortactin (H-191), p-cortactin (Tyr466), paxillin (T-16), ppaxillin (Tyr118), E-cadherin (G-10), vimentin (E-5), actin (C11) (Santa Cruz Biotechnology); α-Tubulin (T9026) (Sigma Aldrich); N-WASP (30D10) (Cell Signaling Technology); p-NWASP (Ser484/485) (Chemicon International); p-Arp2 (Thr237) (Biorbyt).

Techniques: Incubation, Inhibition

Journal: Frontiers in endocrinology

Article Title: Molecular Actions of Thyroid Hormone on Breast Cancer Cell Migration and Invasion via Cortactin/N-WASP.

doi: 10.3389/fendo.2019.00139

Figure Lengend Snippet: FIGURE 6 | Schematic signaling cascade triggered by T3 promoting BC cell migration and invasion. Binding of T3 to the specific membrane integrin αvβ3 receptor induces phosphorylation of the Src/FAK/paxillin/cortactin/N-WASP/ Arp2/3 Complex cascade, promoting BC cell motility.

Article Snippet: Antibodies used were: p-FAKY397 (611807), FAK (610088) (BD Transduction Laboratories, Lexington, KY); p-FAK (Tyr397) (sc-11765-R), cortactin (H-191), p-cortactin (Tyr466), paxillin (T-16), ppaxillin (Tyr118), E-cadherin (G-10), vimentin (E-5), actin (C11) (Santa Cruz Biotechnology); α-Tubulin (T9026) (Sigma Aldrich); N-WASP (30D10) (Cell Signaling Technology); p-NWASP (Ser484/485) (Chemicon International); p-Arp2 (Thr237) (Biorbyt).

Techniques: Migration, Binding Assay, Membrane, Phospho-proteomics