Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Impact of NRP-1 and RhoA in tumor formation and p38 signaling. A and B) Spheroid-derived SCC-13 and SCC-13-NRP-1-KOc8 cells were injected (100 000 cells per each front flank) into NSG mice. Tumors were harvested at 4 wk and extracts prepared for immunoblot. C and D) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg EG00229/kg body weight. Four week tumors were harvested, photographed and extracts were assayed by immunoblot. E–G) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg Y27632/kg body weight. Tumors were harvested at 4 wk, photographed, and assayed by immunoblot. K) Proposed VEGF-A/NRP-1/RhoA/p38 signal transduction pathway. The VEGF-A/NRP-1 complex formation causes the NRP-1 PDZ binding domain to engage the PDZ domain of one subunit of the GIPC1 dimer while the second GIPC1 subunit engages the Syx PDZ binding domain. Syx then activates RhoA which interacts with ROCK to stimulate MAPK signaling. Although MEK3/6 is shown as an activator of p38 activity, this role appears to be context-dependent and we cannot rule out a role for other p38 activation mechanisms. The plotted values are mean ± SEM. The asterisks in each plot indicate a significant reducing in tumor volume, n = 10 tumors/group, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Derivative Assay, Injection, Western Blot, Transduction, Binding Assay, Activity Assay, Activation Assay

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Role of NRP-1 and RhoA in HaCaT ECS cells. A and B) Cells, electroporated with 3 μg control- or NRP-1-siRNA, were tested for spheroid formation and extracts were prepared to assay NRP-1 and MEK3/6 and p38. C) Cells were electroporated with control- or NRP-1- siRNA. After 48 h, extracts were prepared to monitor RhoA level and RhoA activity. D–F) Cells were electroporated with 3 μg of control- or GIPC1-siRNA and tested for spheroid formation, invasion, and MAPK signaling. G–I) Cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation, invasive potential, and MAPK signaling. J–L) Cells were electroporated with 3 μg of control- or NRP1-siRNA in the presence of empty vector (EV) or RhoA WT expression plasmid, and the impact on spheroid formation (3 d) and p38 activity was monitored. All graphical data is presented as the mean ± SEM. In panels A, D, and E, the single asterisk indicates a significant reduction in the treated group compared to control, n = 3, P ≤ 0.005. In panel J the asterisk indicates a significant reduction in spheroid formation in NRP1-siRNA treated versus control. The double asterisk indicates a significant increase in spheroid formation in NRP-1 knockout cells electroporated with RhoA WT expression plasmid versus cells electroporated with empty vector (EV), n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Plasmid Preparation, Expressing, Knock-Out

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: NRP-1 is required for ECS cell survival. A–D) Wild-type and NRP-1 knockdown SCC-13 derived ECS cells were assayed for ability to form spheroids, invade matrigel and migrate. Cells were seeded and grown as spheroids in ultra-low attachment plates. Spheroid diameter and number were measured at 5 d. For invasion, 25 000 cells were seeded atop matrigel per transwell chamber, and cell migration to the lower chamber was monitored at 24 h. For migration, cells were permitted to attach to conventional plates at confluence, wounded and wound closure was monitored. Bars = 100 μm. E) ECS cell lysates were prepared from 5 d spheroids and 300 μg of protein extract was tested for ability to enhanced HUVEC tube formation. The plot shows the number of junctions, segments, and nodes measured using ImageJ analysis.9 The asterisks indicate a significant change (n = 3, P ≤ 0.005). F) NRP-1 interacts with GIPC1, Syx and p38. Extracts were prepared from SCC-13-derived ECS cells and 200 μg of extract was immunoprecipitated with anti-NRP-1 followed by immunoblot to detect the indicated epitopes. Total extract (TE) was electrophoresed as a loading control. G-I) GIPC1 and Syx knockdown reduces spheroid formation and invasion. Cells were electroporated with the indicated siRNA and spheroid number (5 d) and matrigel invasion (24 h) were monitored. J) Cell lysates were prepared from 5 d spheroids and 300 μg protein was tested for ability to enhance HUVEC cell tube formation. The number of junctions, segments, and nodes was assessed using ImageJ.9 All graphical data is presented as the mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Knockdown, Derivative Assay, Migration, Immunoprecipitation, Western Blot, Control

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: RhoA is required for the ECS cell phenotype. A and B) SCC-13 cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation and invasive potential. C and D) Spheroid formation and invasive potential were monitored after treatment with 0 or 20 μM Y27632, a ROCK inhibitor. E) Wild-type and NRP-1 knockout ECS cell extracts were monitored for signaling protein activity. F) SCC-13 cells were electroporated with 3 µg of control- or VEGFA-siRNA, grown as spheroids, and at 5 d assayed for the indicated proteins. G) SCC13 cells were grown as spheroids for 3 d and then treated for 48 h with 0 or 100 μM EG00229 before extracts were prepared for immunoblot. H-J) SCC-13 ECS cells were electroporated with 3 µg of control-, GIPC1-, Syx-, or RhoA-siRNA and impact on MAPK signaling was assayed. K) SCC13 ECS cells were grown as spheroids for 3 d prior to treatment for 48 h with 0 or 20 μM Y27632 and p38 activity was measured. L) SCC-13 ECS cells were electroporated with control- or NRP-1-siRNA and after 48 h extracts were prepared to monitor RhoA level and activity. Similar results were observed in three experiments. The bars = 100 μm. All graphical data is presented as mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Knock-Out, Activity Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Impact of NRP-1 and RhoA in tumor formation and p38 signaling. A and B) Spheroid-derived SCC-13 and SCC-13-NRP-1-KOc8 cells were injected (100 000 cells per each front flank) into NSG mice. Tumors were harvested at 4 wk and extracts prepared for immunoblot. C and D) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg EG00229/kg body weight. Four week tumors were harvested, photographed and extracts were assayed by immunoblot. E–G) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg Y27632/kg body weight. Tumors were harvested at 4 wk, photographed, and assayed by immunoblot. K) Proposed VEGF-A/NRP-1/RhoA/p38 signal transduction pathway. The VEGF-A/NRP-1 complex formation causes the NRP-1 PDZ binding domain to engage the PDZ domain of one subunit of the GIPC1 dimer while the second GIPC1 subunit engages the Syx PDZ binding domain. Syx then activates RhoA which interacts with ROCK to stimulate MAPK signaling. Although MEK3/6 is shown as an activator of p38 activity, this role appears to be context-dependent and we cannot rule out a role for other p38 activation mechanisms. The plotted values are mean ± SEM. The asterisks in each plot indicate a significant reducing in tumor volume, n = 10 tumors/group, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Derivative Assay, Injection, Western Blot, Transduction, Binding Assay, Activity Assay, Activation Assay

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Role of NRP-1 and RhoA in HaCaT ECS cells. A and B) Cells, electroporated with 3 μg control- or NRP-1-siRNA, were tested for spheroid formation and extracts were prepared to assay NRP-1 and MEK3/6 and p38. C) Cells were electroporated with control- or NRP-1- siRNA. After 48 h, extracts were prepared to monitor RhoA level and RhoA activity. D–F) Cells were electroporated with 3 μg of control- or GIPC1-siRNA and tested for spheroid formation, invasion, and MAPK signaling. G–I) Cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation, invasive potential, and MAPK signaling. J–L) Cells were electroporated with 3 μg of control- or NRP1-siRNA in the presence of empty vector (EV) or RhoA WT expression plasmid, and the impact on spheroid formation (3 d) and p38 activity was monitored. All graphical data is presented as the mean ± SEM. In panels A, D, and E, the single asterisk indicates a significant reduction in the treated group compared to control, n = 3, P ≤ 0.005. In panel J the asterisk indicates a significant reduction in spheroid formation in NRP1-siRNA treated versus control. The double asterisk indicates a significant increase in spheroid formation in NRP-1 knockout cells electroporated with RhoA WT expression plasmid versus cells electroporated with empty vector (EV), n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Plasmid Preparation, Expressing, Knock-Out

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: NRP-1 is required for ECS cell survival. A–D) Wild-type and NRP-1 knockdown SCC-13 derived ECS cells were assayed for ability to form spheroids, invade matrigel and migrate. Cells were seeded and grown as spheroids in ultra-low attachment plates. Spheroid diameter and number were measured at 5 d. For invasion, 25 000 cells were seeded atop matrigel per transwell chamber, and cell migration to the lower chamber was monitored at 24 h. For migration, cells were permitted to attach to conventional plates at confluence, wounded and wound closure was monitored. Bars = 100 μm. E) ECS cell lysates were prepared from 5 d spheroids and 300 μg of protein extract was tested for ability to enhanced HUVEC tube formation. The plot shows the number of junctions, segments, and nodes measured using ImageJ analysis.9 The asterisks indicate a significant change (n = 3, P ≤ 0.005). F) NRP-1 interacts with GIPC1, Syx and p38. Extracts were prepared from SCC-13-derived ECS cells and 200 μg of extract was immunoprecipitated with anti-NRP-1 followed by immunoblot to detect the indicated epitopes. Total extract (TE) was electrophoresed as a loading control. G-I) GIPC1 and Syx knockdown reduces spheroid formation and invasion. Cells were electroporated with the indicated siRNA and spheroid number (5 d) and matrigel invasion (24 h) were monitored. J) Cell lysates were prepared from 5 d spheroids and 300 μg protein was tested for ability to enhance HUVEC cell tube formation. The number of junctions, segments, and nodes was assessed using ImageJ.9 All graphical data is presented as the mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Knockdown, Derivative Assay, Migration, Immunoprecipitation, Western Blot, Control

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: RhoA is required for the ECS cell phenotype. A and B) SCC-13 cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation and invasive potential. C and D) Spheroid formation and invasive potential were monitored after treatment with 0 or 20 μM Y27632, a ROCK inhibitor. E) Wild-type and NRP-1 knockout ECS cell extracts were monitored for signaling protein activity. F) SCC-13 cells were electroporated with 3 µg of control- or VEGFA-siRNA, grown as spheroids, and at 5 d assayed for the indicated proteins. G) SCC13 cells were grown as spheroids for 3 d and then treated for 48 h with 0 or 100 μM EG00229 before extracts were prepared for immunoblot. H-J) SCC-13 ECS cells were electroporated with 3 µg of control-, GIPC1-, Syx-, or RhoA-siRNA and impact on MAPK signaling was assayed. K) SCC13 ECS cells were grown as spheroids for 3 d prior to treatment for 48 h with 0 or 20 μM Y27632 and p38 activity was measured. L) SCC-13 ECS cells were electroporated with control- or NRP-1-siRNA and after 48 h extracts were prepared to monitor RhoA level and activity. Similar results were observed in three experiments. The bars = 100 μm. All graphical data is presented as mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Knock-Out, Activity Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Impact of NRP-1 and RhoA in tumor formation and p38 signaling. A and B) Spheroid-derived SCC-13 and SCC-13-NRP-1-KOc8 cells were injected (100 000 cells per each front flank) into NSG mice. Tumors were harvested at 4 wk and extracts prepared for immunoblot. C and D) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg EG00229/kg body weight. Four week tumors were harvested, photographed and extracts were assayed by immunoblot. E–G) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg Y27632/kg body weight. Tumors were harvested at 4 wk, photographed, and assayed by immunoblot. K) Proposed VEGF-A/NRP-1/RhoA/p38 signal transduction pathway. The VEGF-A/NRP-1 complex formation causes the NRP-1 PDZ binding domain to engage the PDZ domain of one subunit of the GIPC1 dimer while the second GIPC1 subunit engages the Syx PDZ binding domain. Syx then activates RhoA which interacts with ROCK to stimulate MAPK signaling. Although MEK3/6 is shown as an activator of p38 activity, this role appears to be context-dependent and we cannot rule out a role for other p38 activation mechanisms. The plotted values are mean ± SEM. The asterisks in each plot indicate a significant reducing in tumor volume, n = 10 tumors/group, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Derivative Assay, Injection, Western Blot, Transduction, Binding Assay, Activity Assay, Activation Assay

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Role of NRP-1 and RhoA in HaCaT ECS cells. A and B) Cells, electroporated with 3 μg control- or NRP-1-siRNA, were tested for spheroid formation and extracts were prepared to assay NRP-1 and MEK3/6 and p38. C) Cells were electroporated with control- or NRP-1- siRNA. After 48 h, extracts were prepared to monitor RhoA level and RhoA activity. D–F) Cells were electroporated with 3 μg of control- or GIPC1-siRNA and tested for spheroid formation, invasion, and MAPK signaling. G–I) Cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation, invasive potential, and MAPK signaling. J–L) Cells were electroporated with 3 μg of control- or NRP1-siRNA in the presence of empty vector (EV) or RhoA WT expression plasmid, and the impact on spheroid formation (3 d) and p38 activity was monitored. All graphical data is presented as the mean ± SEM. In panels A, D, and E, the single asterisk indicates a significant reduction in the treated group compared to control, n = 3, P ≤ 0.005. In panel J the asterisk indicates a significant reduction in spheroid formation in NRP1-siRNA treated versus control. The double asterisk indicates a significant increase in spheroid formation in NRP-1 knockout cells electroporated with RhoA WT expression plasmid versus cells electroporated with empty vector (EV), n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Plasmid Preparation, Expressing, Knock-Out

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: NRP-1 is required for ECS cell survival. A–D) Wild-type and NRP-1 knockdown SCC-13 derived ECS cells were assayed for ability to form spheroids, invade matrigel and migrate. Cells were seeded and grown as spheroids in ultra-low attachment plates. Spheroid diameter and number were measured at 5 d. For invasion, 25 000 cells were seeded atop matrigel per transwell chamber, and cell migration to the lower chamber was monitored at 24 h. For migration, cells were permitted to attach to conventional plates at confluence, wounded and wound closure was monitored. Bars = 100 μm. E) ECS cell lysates were prepared from 5 d spheroids and 300 μg of protein extract was tested for ability to enhanced HUVEC tube formation. The plot shows the number of junctions, segments, and nodes measured using ImageJ analysis.9 The asterisks indicate a significant change (n = 3, P ≤ 0.005). F) NRP-1 interacts with GIPC1, Syx and p38. Extracts were prepared from SCC-13-derived ECS cells and 200 μg of extract was immunoprecipitated with anti-NRP-1 followed by immunoblot to detect the indicated epitopes. Total extract (TE) was electrophoresed as a loading control. G-I) GIPC1 and Syx knockdown reduces spheroid formation and invasion. Cells were electroporated with the indicated siRNA and spheroid number (5 d) and matrigel invasion (24 h) were monitored. J) Cell lysates were prepared from 5 d spheroids and 300 μg protein was tested for ability to enhance HUVEC cell tube formation. The number of junctions, segments, and nodes was assessed using ImageJ.9 All graphical data is presented as the mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Knockdown, Derivative Assay, Migration, Immunoprecipitation, Western Blot, Control

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: RhoA is required for the ECS cell phenotype. A and B) SCC-13 cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation and invasive potential. C and D) Spheroid formation and invasive potential were monitored after treatment with 0 or 20 μM Y27632, a ROCK inhibitor. E) Wild-type and NRP-1 knockout ECS cell extracts were monitored for signaling protein activity. F) SCC-13 cells were electroporated with 3 µg of control- or VEGFA-siRNA, grown as spheroids, and at 5 d assayed for the indicated proteins. G) SCC13 cells were grown as spheroids for 3 d and then treated for 48 h with 0 or 100 μM EG00229 before extracts were prepared for immunoblot. H-J) SCC-13 ECS cells were electroporated with 3 µg of control-, GIPC1-, Syx-, or RhoA-siRNA and impact on MAPK signaling was assayed. K) SCC13 ECS cells were grown as spheroids for 3 d prior to treatment for 48 h with 0 or 20 μM Y27632 and p38 activity was measured. L) SCC-13 ECS cells were electroporated with control- or NRP-1-siRNA and after 48 h extracts were prepared to monitor RhoA level and activity. Similar results were observed in three experiments. The bars = 100 μm. All graphical data is presented as mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Knock-Out, Activity Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Impact of NRP-1 and RhoA in tumor formation and p38 signaling. A and B) Spheroid-derived SCC-13 and SCC-13-NRP-1-KOc8 cells were injected (100 000 cells per each front flank) into NSG mice. Tumors were harvested at 4 wk and extracts prepared for immunoblot. C and D) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg EG00229/kg body weight. Four week tumors were harvested, photographed and extracts were assayed by immunoblot. E–G) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg Y27632/kg body weight. Tumors were harvested at 4 wk, photographed, and assayed by immunoblot. K) Proposed VEGF-A/NRP-1/RhoA/p38 signal transduction pathway. The VEGF-A/NRP-1 complex formation causes the NRP-1 PDZ binding domain to engage the PDZ domain of one subunit of the GIPC1 dimer while the second GIPC1 subunit engages the Syx PDZ binding domain. Syx then activates RhoA which interacts with ROCK to stimulate MAPK signaling. Although MEK3/6 is shown as an activator of p38 activity, this role appears to be context-dependent and we cannot rule out a role for other p38 activation mechanisms. The plotted values are mean ± SEM. The asterisks in each plot indicate a significant reducing in tumor volume, n = 10 tumors/group, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Derivative Assay, Injection, Western Blot, Transduction, Binding Assay, Activity Assay, Activation Assay

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Role of NRP-1 and RhoA in HaCaT ECS cells. A and B) Cells, electroporated with 3 μg control- or NRP-1-siRNA, were tested for spheroid formation and extracts were prepared to assay NRP-1 and MEK3/6 and p38. C) Cells were electroporated with control- or NRP-1- siRNA. After 48 h, extracts were prepared to monitor RhoA level and RhoA activity. D–F) Cells were electroporated with 3 μg of control- or GIPC1-siRNA and tested for spheroid formation, invasion, and MAPK signaling. G–I) Cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation, invasive potential, and MAPK signaling. J–L) Cells were electroporated with 3 μg of control- or NRP1-siRNA in the presence of empty vector (EV) or RhoA WT expression plasmid, and the impact on spheroid formation (3 d) and p38 activity was monitored. All graphical data is presented as the mean ± SEM. In panels A, D, and E, the single asterisk indicates a significant reduction in the treated group compared to control, n = 3, P ≤ 0.005. In panel J the asterisk indicates a significant reduction in spheroid formation in NRP1-siRNA treated versus control. The double asterisk indicates a significant increase in spheroid formation in NRP-1 knockout cells electroporated with RhoA WT expression plasmid versus cells electroporated with empty vector (EV), n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Plasmid Preparation, Expressing, Knock-Out

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: NRP-1 is required for ECS cell survival. A–D) Wild-type and NRP-1 knockdown SCC-13 derived ECS cells were assayed for ability to form spheroids, invade matrigel and migrate. Cells were seeded and grown as spheroids in ultra-low attachment plates. Spheroid diameter and number were measured at 5 d. For invasion, 25 000 cells were seeded atop matrigel per transwell chamber, and cell migration to the lower chamber was monitored at 24 h. For migration, cells were permitted to attach to conventional plates at confluence, wounded and wound closure was monitored. Bars = 100 μm. E) ECS cell lysates were prepared from 5 d spheroids and 300 μg of protein extract was tested for ability to enhanced HUVEC tube formation. The plot shows the number of junctions, segments, and nodes measured using ImageJ analysis.9 The asterisks indicate a significant change (n = 3, P ≤ 0.005). F) NRP-1 interacts with GIPC1, Syx and p38. Extracts were prepared from SCC-13-derived ECS cells and 200 μg of extract was immunoprecipitated with anti-NRP-1 followed by immunoblot to detect the indicated epitopes. Total extract (TE) was electrophoresed as a loading control. G-I) GIPC1 and Syx knockdown reduces spheroid formation and invasion. Cells were electroporated with the indicated siRNA and spheroid number (5 d) and matrigel invasion (24 h) were monitored. J) Cell lysates were prepared from 5 d spheroids and 300 μg protein was tested for ability to enhance HUVEC cell tube formation. The number of junctions, segments, and nodes was assessed using ImageJ.9 All graphical data is presented as the mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Knockdown, Derivative Assay, Migration, Immunoprecipitation, Western Blot, Control

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: RhoA is required for the ECS cell phenotype. A and B) SCC-13 cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation and invasive potential. C and D) Spheroid formation and invasive potential were monitored after treatment with 0 or 20 μM Y27632, a ROCK inhibitor. E) Wild-type and NRP-1 knockout ECS cell extracts were monitored for signaling protein activity. F) SCC-13 cells were electroporated with 3 µg of control- or VEGFA-siRNA, grown as spheroids, and at 5 d assayed for the indicated proteins. G) SCC13 cells were grown as spheroids for 3 d and then treated for 48 h with 0 or 100 μM EG00229 before extracts were prepared for immunoblot. H-J) SCC-13 ECS cells were electroporated with 3 µg of control-, GIPC1-, Syx-, or RhoA-siRNA and impact on MAPK signaling was assayed. K) SCC13 ECS cells were grown as spheroids for 3 d prior to treatment for 48 h with 0 or 20 μM Y27632 and p38 activity was measured. L) SCC-13 ECS cells were electroporated with control- or NRP-1-siRNA and after 48 h extracts were prepared to monitor RhoA level and activity. Similar results were observed in three experiments. The bars = 100 μm. All graphical data is presented as mean ± SEM and the asterisks indicate a significant change, n = 3, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Knock-Out, Activity Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Impact of NRP-1 and RhoA in tumor formation and p38 signaling. A and B) Spheroid-derived SCC-13 and SCC-13-NRP-1-KOc8 cells were injected (100 000 cells per each front flank) into NSG mice. Tumors were harvested at 4 wk and extracts prepared for immunoblot. C and D) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg EG00229/kg body weight. Four week tumors were harvested, photographed and extracts were assayed by immunoblot. E–G) Spheroid-derived SCC-13 cells were injected (100 000 per each front flank) into NSG mice and treated with 0 or 10 mg Y27632/kg body weight. Tumors were harvested at 4 wk, photographed, and assayed by immunoblot. K) Proposed VEGF-A/NRP-1/RhoA/p38 signal transduction pathway. The VEGF-A/NRP-1 complex formation causes the NRP-1 PDZ binding domain to engage the PDZ domain of one subunit of the GIPC1 dimer while the second GIPC1 subunit engages the Syx PDZ binding domain. Syx then activates RhoA which interacts with ROCK to stimulate MAPK signaling. Although MEK3/6 is shown as an activator of p38 activity, this role appears to be context-dependent and we cannot rule out a role for other p38 activation mechanisms. The plotted values are mean ± SEM. The asterisks in each plot indicate a significant reducing in tumor volume, n = 10 tumors/group, P ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Derivative Assay, Injection, Western Blot, Transduction, Binding Assay, Activity Assay, Activation Assay

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: Role of NRP-1 and RhoA in HaCaT ECS cells. A and B) Cells, electroporated with 3 μg control- or NRP-1-siRNA, were tested for spheroid formation and extracts were prepared to assay NRP-1 and MEK3/6 and p38. C) Cells were electroporated with control- or NRP-1- siRNA. After 48 h, extracts were prepared to monitor RhoA level and RhoA activity. D–F) Cells were electroporated with 3 μg of control- or GIPC1-siRNA and tested for spheroid formation, invasion, and MAPK signaling. G–I) Cells were electroporated with 3 μg of control- or RhoA-siRNA and tested for spheroid formation, invasive potential, and MAPK signaling. J–L) Cells were electroporated with 3 μg of control- or NRP1-siRNA in the presence of empty vector (EV) or RhoA WT expression plasmid, and the impact on spheroid formation (3 d) and p38 activity was monitored. All graphical data is presented as the mean ± SEM. In panels A, D, and E, the single asterisk indicates a significant reduction in the treated group compared to control, n = 3, P ≤ 0.005. In panel J the asterisk indicates a significant reduction in spheroid formation in NRP1-siRNA treated versus control. The double asterisk indicates a significant increase in spheroid formation in NRP-1 knockout cells electroporated with RhoA WT expression plasmid versus cells electroporated with empty vector (EV), n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Plasmid Preparation, Expressing, Knock-Out

Journal: Molecular carcinogenesis

Article Title: NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survival

doi: 10.1002/mc.22943

Figure Lengend Snippet: The ECS cell phenotype and MEK3/6 and p38 signaling. A-C) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA and the impact on p38 activity, and spheroid formation and invasive, were measured. D) Cells were electroporated with 3 μg of Control-, MEK3-, or MEK6-siRNA, grown for 5 d as spheroids, and cell extracts (300 μg protein) were assayed for ability to enhance HUVEC tube formation as measured by junction, segment and node formation. E) Cells were electroporated with 3 μg of the indicated siRNA and p38 level and activity were measured at 48 h. F and G) Impact of p38 knockdown on spheroid formation and matrigel invasion. H and I) Treatment with SB203580, a p38α/β inhibitor, suppresses ECS cell spheroid formation and invasive potential. J and K) RhoA restores the ECS cell phenotype in NRP-1 knockdown cells. SCC-13 derived NRP-1 knockout cells were electroporated with 3 μg of empty vector or RhoA WT expression plasmid, and plated for spheroid formation (5 d). The immunoblot monitors NRP-1 knockdown, RhoA overexpression, and the impact on p38T and p38-P level. SCC-13 and SCC13-NRP-1-KOc8 cells were electroporated with 3 μg of the indicated plasmids and ability to form spheroids was monitored at 5 d. The asterisk indicates a significant reduction in spheroid formation in SCC13-NRP1-KOc8 cells versus SCC-13. The double asterisk indicates an increase in spheroid formation in RhoA WT expression plasmid-electroporated versus empty vector-electroporated SCC13-NRP1-KOc8 cells, n = 3, P ≤ 0.005. All bars = 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: For electroporation, 1 × 10 6 cells were electroporated with 3 μg of control- (D-001206–13-05, Dharmacon), VEGF-A (sc-29520, Santa Cruz), NRP-1 (sc-36038, Santa Cruz), GIPC1 (M-019997–02-005, Dharmacon), p38α (sc-29433, Santa Cruz), p38β (sc-39116, Santa Cruz), p38γ (sc-39118, Santa Cruz), p38δ (sc-36456, Santa Cruz), MEKK1 (sc-35898, Santa Cruz), MEK3 (sc-35907, Santa Cruz), MEK6 (sc-35913, Santa Cruz), Syx (Dharmacon, M-013873–01-0005) or RhoA (Santa Cruz, sc-29471) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany).

Techniques: Control, Activity Assay, Knockdown, Derivative Assay, Knock-Out, Plasmid Preparation, Expressing, Western Blot, Over Expression