Journal: Journal of Cell Science

Article Title: The gap junction protein connexin 43 controls multiple aspects of cranial neural crest cell development

doi: 10.1242/jcs.235440

Figure Lengend Snippet: Cx43 depletion decreases the number of premigratory neural crest cells in the absence of any changes in cell proliferation or cell death. Representative transverse sections taken through the midbrain and hindbrain of HH8+ to HH9 embryos after unilateral electroporation of the neural tube at HH7+ to HH8− (2ss–3ss) with either the control morpholino (MM) or Cx43 MO and immunostaining for various neural crest cell markers. In MM-treated embryos (A–A″,C–C″,E–E″,G–G″,I–I″,K–K″,M–M″), there is no decrease in the level of Cx43 in the neural tube (A′) and no change in any of the premigratory neural crest cells as identified by Pax7 (C′), Snail2 (E′), Sox10 (G′). Similarly, there are no changes in N-cadherin (I–I″), a marker of neural tube cells, cell proliferation [pHH3 (K–K″)]; or cell death [TUNEL (M–M″)]. In Cx43 MO-treated embryos (B–B″,D–D″,F–F″,H–H″,J–J″,L–L″,N–N″), however, there is obvious knockdown of Cx43 in the neural tube (B′, arrow) and also a reduction in the premigratory neural crest cell population observed after Pax7 (D′, arrow), Snail2 (F′, arrow) and Sox10 (H′, arrow) immunostaining. Interestingly, N-cadherin (J–J″) is not affected. Cx43 MO-mediated loss of Cx43 also does not seem to alter levels of either cell proliferation (L–L″) or cell death (N–N″). Additionally, in HH9 embryos, a small number of MO-positive neural crest cells begin to emerge from the neural tube (D′,F′,H′, arrowheads; neural tube indicated by dashed lines). Scale bars: 20 µm (A–K″,M–N″); 50 µm (L–L″).

Article Snippet: The following day, after washing four times for 30 min with 1× PBST, sections were incubated with appropriate secondary antibodies and incubated for 2–3 h at room temperature or overnight at 4°C [goat anti-rabbit IgG 488 or 647; Invitrogen, A11034 and Jackson ImmunoResearch, PA, USA; 111-605-003, respectively (for Cx43, Sox10, pHH3 and Snail2); goat anti-mouse IgG 1 647; Invitrogen, A21240 (for Pax7 and E-cadherin); goat anti-rat IgG; Life Technologies, MD; A21247 (for N-cadherin)].

Techniques: Electroporation, Control, Immunostaining, Marker, TUNEL Assay, Knockdown

Journal: Journal of Cell Science

Article Title: The gap junction protein connexin 43 controls multiple aspects of cranial neural crest cell development

doi: 10.1242/jcs.235440

Figure Lengend Snippet: Graphical representation of analyzed premigratory neural crest cell data after control or Cx43 MO treatment. Box and whisker plots showing the third quartile (Q3), median and first quartile (Q1) range of the data as well as data outliers. Whiskers represent minimum and maximum values. (A–C) Graphs depicting the number of premigratory, migratory and total neural crest cells (NCCs) electroporated with either the control MM or Cx43 MO normalized to the un-electroporated contralateral side for the analyzed neural crest cell markers Pax7 (A), Snail2 (B) and Sox10 (C). Asterisks denote statistically significant differences determined with un-paired Mann–Whitney (Wilcoxon) test that employed a Bonferroni correction [A, Cx43 MO premigratory NCCs (P=8.64×10−7); B, Cx43 MO premigratory NCCs (P=6.41×10−9), Cx43 MO migratory NCCs (P=0.0029), Cx43 MO Total NCCs (P=9.64×10−9); C, Cx43 MO Premigratory NCCs (P=0.0001), Cx43 MO Total NCCs (P=2.76×10−6)]. (D) Graph depicting the fluorescence intensity of N-cadherin in premigratory neural crest cells (NCCs) and the neural tube (NT) of control MM- (N=4) or Cx43 MO- (N=5) electroporated cells compared to the contralateral side.

Article Snippet: The following day, after washing four times for 30 min with 1× PBST, sections were incubated with appropriate secondary antibodies and incubated for 2–3 h at room temperature or overnight at 4°C [goat anti-rabbit IgG 488 or 647; Invitrogen, A11034 and Jackson ImmunoResearch, PA, USA; 111-605-003, respectively (for Cx43, Sox10, pHH3 and Snail2); goat anti-mouse IgG 1 647; Invitrogen, A21240 (for Pax7 and E-cadherin); goat anti-rat IgG; Life Technologies, MD; A21247 (for N-cadherin)].

Techniques: Control, Whisker Assay, MANN-WHITNEY, Fluorescence

Journal: Oncotarget

Article Title: PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

doi: 10.18632/oncotarget.6640

Figure Lengend Snippet: A. Migration and invasion assay in GBM cells transfected with control or PKC isoform siRNAs as indicated. B. Migration and invasion assay in GBM cells transfected with control or PKCδ siRNAs. C. Effect of PKCδ depletion on infiltration of U87 GBM cells in collagen-based matrix three-dimensional (3D) culture system. Scale bar, 100 μm. D. Western blot analysis for mesenchymal markers and regulators in U87 GBM cells transfected with control or PKCδ siRNAs. E. Immunocytochemistry for CDH2 and VIM in U87 GBM cells transfected with control or PKCδ siRNAs. F. Migration and invasion assay in GBM cells transduced with MFG or HA-PKCδ. G. Effect of PKCδ on infiltration of U87 GBM cells in collagen-based matrix 3D culture system. Scale bar, 100 μm. H. Western blot analysis for mesenchymal markers and regulators in U87 GBM cells transduced with MFG or HA-PKCδ. I. Immunocytochemistry for CDH2 and VIM in U87 GBM cells transduced with MFG or HA-PKCδ. J, K. Immunohistochemistry for p-PKCδ (J), and CDH2, VIM (K) in orthotopic U87 cell-xenograft tumors. U87 GBM cells were transduced with pSuper or PKCδ shRNA prior to injection to mice. Scale bar, 200 μm. L, M. q-RT PCR (L) and Western blot analysis (M) for mesenchymal markers and regulators in the orthotopic xenograft tumors. β-actin was used for a loading control. *, P < 0.05 versus control; **, p <0.01 versus control.

Article Snippet: Polyclonal antibodies to PKCδ, vimentin (VIM), TWIST11, SNAI1 (also known as SNAI1), SNAI2 (also known as SLUG), STAT3, SRC, IκB, NFκB, Jagged-1 (JAG1), Jagged-2 (JAG2) and CDH2 (also known as N-cadherin) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Migration, Invasion Assay, Transfection, Control, Western Blot, Immunocytochemistry, Transduction, Immunohistochemistry, shRNA, Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

doi: 10.18632/oncotarget.6640

Figure Lengend Snippet: A, B. Western blot analysis for phosphorylation status of SRC and STAT3 in GBM cells transfected with control or PKCδ siRNAs (A), or transduced with MFG or HA-tagged PKCδ (B). C, D. Migration and invasion assay in GBM cells transfected with control or SRC (C), or STAT3 siRNAs (D). E. Infiltration of GBM cells transfected with control siRNAs or siRNAs against SRC or STAT3 in collagen-based matrix 3D culture system. Scale bar, 100 μm. F, G. Immunohistochemistry (F) and western blot analysis (G) for p-SRC and p-STAT3 in orthotopic U87 GBM cell-xenograft tumors. U87 GBM cells were transduced with control or PKCδ shRNA prior to orthotopic injection to mice. Scale bar, 200 μm. (H, I) Western blot analysis for CDH2, SNAI2 and ZEB1 H. , and immunocytochemistry for CDH2 I. in U87 GBM transfected with control or SRC siRNAs. (J, K) Western blot analysis for CDH2, SNAI2 and ZEB1 J. , and immunocytochemistry for CDH2 K. in U87 GBM cells transfected with control or STAT3 siRNAs. L. Western blot analysis for p-STAT3 in U87 GBM cells transfected with control or SRC siRNAs. M. Western blot analysis for p-SRC in U87 GBM cells transfected with control or STAT3 siRNAs. β-actin was used for a loading control. *, P < 0.05 versus control; **, p <0.01 versus control.

Article Snippet: Polyclonal antibodies to PKCδ, vimentin (VIM), TWIST11, SNAI1 (also known as SNAI1), SNAI2 (also known as SLUG), STAT3, SRC, IκB, NFκB, Jagged-1 (JAG1), Jagged-2 (JAG2) and CDH2 (also known as N-cadherin) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Phospho-proteomics, Transfection, Control, Transduction, Migration, Invasion Assay, Immunohistochemistry, shRNA, Injection, Immunocytochemistry

Journal: Oncotarget

Article Title: PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

doi: 10.18632/oncotarget.6640

Figure Lengend Snippet: A, B. qRT-PCR for NOTCH isoforms (A) and the ligands (B) in U87 GBM cells transfected with control or PKCδ siRNAs. C, D. Immunocytochemistry for NOTCH2 (C) and ligands JAG1 and -2 (D) in U87 GBM cells transfected with control or PKCδ siRNAs. E. Western blot analysis for NOTCH2 and its ligands JAG1 and -2 in U87 GBM cells transduced with MFG or HA-PKCδ. F, G. Migration and invasion assay in U87 GBM cells transfected with NOTCH2 siRNAs (F) or treated with γ-secretase inhibitor (GSI) (G), as compared to control. H. Infiltration of U87 GBM cells transfected with control or NOTCH2 siRNAs in collagen-based matrix 3D culture system. Scale bar, 100 μm. I. Migration and invasion assay in U87 GBM cells transfected with control siRNAs or siRNAs against JAG1 or -2. J. Western blot analysis for NICD2 in U87 GBM cells transfected with control siRNAs or siRNAs against JAG1 or -2. K, L. Western blot analysis for CDH2, SNAI2 and ZEB1 (K), and immunocytochemistry for CDH2 (L) in U87 GBM cells transfected with NOTCH2 siRNAs or treated with GSI. M, N. Immunohistochemical staining (M) and qRT-PCR (N) for NOTCH-2, JAG1 and -2 in orthotopic xenograft tumors formed by U87 GBM cells transduced with control (pSuper) or PKCδ shRNAs. Scale bar, 200 μm. O, P. qRT-PCR for NOTCH-2, JAG1 and -2 in U87 GBM cells transfected by control siRNAs or siRNAs against SRC (O) or STAT3 (P). Q. Western blot analysis for NICD2 in U87 GBM cells transfected by control siRNAs or siRNAs against SRC or STAT3. β-actin was used for a loading control. *, P < 0.05 versus control; **, p <0.01 versus control.

Article Snippet: Polyclonal antibodies to PKCδ, vimentin (VIM), TWIST11, SNAI1 (also known as SNAI1), SNAI2 (also known as SLUG), STAT3, SRC, IκB, NFκB, Jagged-1 (JAG1), Jagged-2 (JAG2) and CDH2 (also known as N-cadherin) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Quantitative RT-PCR, Transfection, Control, Immunocytochemistry, Western Blot, Transduction, Migration, Invasion Assay, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

doi: 10.18632/oncotarget.6640

Figure Lengend Snippet: A. Western blot analysis for activation status of PKCδ in U87 GBM cells transfected with control, c-MET, EGFR or EGFRvIII siRNAs. B. Migration and invasion assay in U87 GBM cells transfected with control or c-MET siRNAs. C. Infiltration of X01 GBM cells transfected with control or c-MET siRNAs in collagen-based matrix 3D culture system. Scale bar, 100 μm. D, E. Western blot analysis for activation status of PKCδ, SRC, STAT3 and NOTCH2 (D) or for CDH2, SNAI2 and ZEB1 (E) in U87 GBM cells transfected with control or c-MET siRNAs. F. qRT-PCR for NOTCH-2, JAG1 and -2 in U87 GBM cells transfected by control or c-MET siRNAs. G. Kinase assay of immunoprecipitated c-MET using GSC-PKCδ as a substrate and western blot analysis for p-PKCδ in U87 GBM cells transfected with control or c-MET siRNAs. β-actin was used for a loading control. *, P < 0.05 versus control; **, p <0.01 versus control.

Article Snippet: Polyclonal antibodies to PKCδ, vimentin (VIM), TWIST11, SNAI1 (also known as SNAI1), SNAI2 (also known as SLUG), STAT3, SRC, IκB, NFκB, Jagged-1 (JAG1), Jagged-2 (JAG2) and CDH2 (also known as N-cadherin) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Activation Assay, Transfection, Control, Migration, Invasion Assay, Quantitative RT-PCR, Kinase Assay, Immunoprecipitation