Journal: Oncotarget

Article Title: Discoidin domain receptor 1 promotes Th17 cell migration by activating the RhoA/ROCK/MAPK/ERK signaling pathway.

doi: 10.18632/oncotarget.10455

Figure Lengend Snippet: Figure 2: DDR1 promotes human Th17 motility in 3D collagen. A. DDR1 expression is reduced by specific DDR1 siRNA (HSS1878780). Polarized Th17 cells were transfected with control or with DDR1 siRNAs and DDR1 expression was assessed by western blot using the anti-DDR1 antibody (C-20). The blot was stripped and reprobed with anti-β-actin antibody to ensure equal loading (top panel). After transfection, the cells were activated with PMA+ionomycin in the presence of brefeldin A to identify IL-17-producing cells, stained with anti-DDR1 and anti-IL-17 antibodies and analyzed by flow cytometry (lower panel). Immunoblot and FACS plots are representative of five independent experiments performed with polarized Th17 cells derived from five different blood donors. B. DDR1 siRNA inhibits Th17 motility in 3D collagen. After transfection, the cells were labelled with calcein AM and embedded in collagen gels. Cell migration was evaluated by live cell confocal microscopy and quantified by computer-assisted cell tracking as described in the “Materials and Methods” section. Representative cell migration tracks over 30 min are presented as x-y projections (distance, in μm) (left panel). The histogram (right panel) represents the mean velocity of 100 cells presented as μm/min. C. DDR1:Fc inhibits Th17 motility in 3D collagen. The cells were embedded in collagen gels containing control human IgG (Fc fragment) or human DDR1:Fc recombinant proteins and cell motility was determined as above. Results (B and C right panels) are mean values ± SD of five independent experiments performed with polarized Th17 cells derived from five different blood donors. *p < 0.05. D. DDR1:Fc inhibits the migratory shape of polarized Th17 cells. Representative photography images from five different experiments of polarized Th17 cells migrating in collagen gels containing either control IgG or DDR1:Fc (400X magnification).

Article Snippet: Non-conjugated rabbit anti-mouse and human DDR1 (clone C-20), non-conjugated rabbit anti-human DDR2 (clone H108) antibodies and the anti-phospho-ERK1/2 (clone E-4), anti-ERK2 (clone C-14), and anti-β-actin (clone C-2) Abs were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Transfection, Control, Western Blot, Staining, Flow Cytometry, Derivative Assay, Migration, Confocal Microscopy, Cell Tracking Assay, Recombinant

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (A) and immunoblotting (B) analysis of VANGL2 mRNA and protein level change in A549 cells infected with vesicular stomatitis virus (VSV) [multiplicity of infection (MOI) of 0.5] for 0 to 16 hours. ( C ) Immunoblotting analysis of VANGL2 protein level changes in wild-type (WT) and Ifnar −/− peritoneal macrophages (PEMs) infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Luciferase reporter assays analyzing IFN-β or IFN-stimulated response element (ISRE) promoter activity of human embryonic kidney (HEK) 293T cells transfected with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 or empty vector (EV) for 24 hours, followed by treatment with or without VSV (MOI of 0.5) (D) or poly(I:C) (E) for 12 hours, respectively. ( F to J ) Immunoblotting analysis (F and I) of total and phosphorylated IRF3 and RT-PCR analysis (G), (H), and (J) of indicated gene expression in HEK293T (F) to (H) or A549 (I) and (J) cells transfected with FLAG-VANGL2 or EV for 24 hours, followed by VSV (MOI of 0.5) infection at indicated time points. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( K and L ) Fluorescence microscopy analysis (K) and flow cytometric analysis (L) of the replication of VSV–enhanced green fluorescent protein (eGFP) in HEK293T cells transfected with EV or increasing HA-VANGL2 at indicated dose for 24 hours, followed by treatment with or without VSV-eGFP (MOI of 0.5) infection at indicated time points. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. NC, negative control. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test. ns, not significant.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Negative Control

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) Luciferase activity in HEK293T cells transfected with scrambled ( Scr ) small interfering RNA (siRNA) or siRNA-targeting VANGL2 for 24 hours and then transfected with an IFN-β luciferase (IFN-β luc) for 24 hours, followed by treatment with or without VSV (MOI of 0.5), poly(I:C), or poly (dA:dT) for 12 hours. ( B and C ) RT-PCR (B) and immunoblotting (C) analysis of VSV (MOI of 0.5)–infected THP-1 cells transfected with Scr siRNA or VANGL2 -specific siRNA at indicated time points. ( D ) Heatmap view of top and bottom gene list of RNA-sequence data sets. Microarray analysis for total RNA was performed for Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + bone marrow–derived macrophages (BMDMs) with or without VSV infection. ( E ) VANGL2 regulates antiviral response-relevant target genes, presented as a volcano plot of genes with differential expression after VSV infection in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs. FC, fold change. ( F ) Gene ontology (GO) enrichment analysis of the VANGL2-dependent genes in (E) (−log 2 P values). ( G ) Heatmap showing the change of indicated ISGs in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs with or without VSV infection. ( H to L ) RT-PCR analysis of Ifnb (H), Isg56 (I), and VSV-G (K) mRNA expression, IFN-β enzyme-linked immunosorbent assay (ELISA) (J), and immunoblotting (L) analysis of total and phosphorylated IRF3 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Sequencing, Microarray, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Luciferase reporter assays analyzing IFN-β (A) or ISRE (B) promoter activity of HEK293T cells transfected with the Flag-tagged indicated plasmids along with EV or increasing amounts (from 100 to 200 ng) of HA-VANGL2. ( C ) Co-immunoprecipitation (co-IP; with anti-FLAG) and immunoblotting analysis using protein lysates of HEK293T cells transfected with indicated plasmids. WCL, whole cell lysates. ( D ) Co-IP (with anti-TBK1) and immunoblotting analysis using endogenous proteins lysates of control and VSV (MOI of 0.5, 12 hours)–infected BMDMs. ( E ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. ( F ) Quantitative analysis of the colocalization in (E). ( G ) Co-IP (with anti-VANGL2) and immunoblotting analysis using unsorted, cytosolic, and membrane lysates of THP-1 cells with or without VSV infection for 12 hours. ( H ) Schematic mapping of VANGL2. ( I ) Co-IP and immunoblotting analysis using lysates from HEK293T cells transfected with MYC-VANGL2 and its truncations along with FLAG-TBK1. ( J ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with vectors for HA-PKBD along with FLAG-TBK1. PkBD, the Prickle-binding domain. ( K and L ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with MYC-VANGL2 and its deletions along with FLAG-TBK1 (K) or infected with VSV (L) (MOI of 0.5). ( M and N ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with increasing amounts (from 300 to 500 ng) of HA-PKBD or EV along with FLAG-TBK1 (M) or infected with VSV (N) (MOI of 0.5). Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection, Labeling, Confocal Microscopy, Binding Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) RT-qPCR (A) and immunoblotting (B) analysis of TBK1 mRNA and protein level change in HEK293T cells transfected with HA-TBK1, and increasing amounts of MYC-VANGL2. ( C ) Immunoblotting analysis of total and phosphorylated TBK1 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Immunoblotting analysis of total and phosphorylated TBK1 using THP-1 (D) or PBMCs (E) transfected with Scr siRNA or VANGL2 siRNA for 24 hours and then infected with VSV (MOI of 0.5) for indicated time. ( F ) Immunoblotting analysis of protein extracts of HEK293T cells treated with CHX (100 μg/ml) for 12 hours, followed by treatment with MG132 (10 μM), Baf A1 (0.2 μM), or both for 6 hours. ( G ) Immunoblotting analysis of HEK293T cells transfected with indicated plasmids for 24 hours, followed by treatment with MG132 (10 μM), 3-MA (10 mM), or Baf A1 (0.2 μM) for 6 hours. ( H ) Immunoblotting analysis using lysates from HEK293T cells transfected with FLAG-VANGL2 or FLAG-EV for 24 hours, followed by treatment with rapamycin (250 nM) for indicated time. ( I ) Immunoblotting analysis using lysates from Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs treated with EBSS for indicated time. ( J ) Immunoblotting analysis of WT, BECLIN -KO, or ATG5 -KO HEK293T cells transfected with indicated plasmids. ( K ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated specific antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; violet, LAMP1 signal; blue, DAPI. Scale bars, 20 μm. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Infection, Labeling, Confocal Microscopy

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with indicated Flag-tagged cargo receptors along with HA-TBK1 (A) or HA-VANGL2 (B). ( C ) Cell lysates were harvested after Baf A1 (0.2 μM) treatment (6 hours) for co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with HA-TBK1, FLAG-OPTN, and MYC-VANGL2. ( D ) Immunoblotting analysis of WT and OPTN −/− HEK293T cells transfected with FLAG-TBK1 and MYC-EV or MYC-VANGL2 for 24 hours. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of WT, OPTN -KO, NDP52- KO, p62 -KO, or TOLLIP -KO HEK293T cells transfected with FLAG-TBK1, together with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 for 24 hours. ( F and G ) A549 (F) or BMDMs (G) cells were infected with VSV (MOI of 0.5), and protein lysates were harvested for IP using an anti-OPTN antibody. ( H to J ) Immunoblotting (H) and RT-PCR (I) and (J) analysis of WT and OPTN -KO HEK293T cells transfected with FLAG-EV or FLAG-VANGL2 for 24 hours, followed by treatment with or without VSV (MOI of 0.5) infection for 12 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** P < 0.001 and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Infection, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with FLAG-TBK1 and HA-tagged WT ubiquitin (HA-Ub) or its mutants, together with MYC-EV or MYC-VANGL2 for 24 hours, followed by treatment with Baf A1 (0.2 μM) for 6 hours, followed by co-immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibody. ( B ) IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( C ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with Scr shRNA or other E3 ligase–specific shRNAs for 24 hours, followed by transfected with FLAG-TBK1, together with EV or HA-VANGL2 for 24 hours. ( D ) HEK293T cells were transfected with Scr shRNA or TRIP- specific shRNA for 24 hours, followed by transfected with FLAG-EV or FLAG-VANGL2 for 24 hours; protein lysates were harvested after VSV (MOI of 0.5) infection for 12 hours and Baf A1 (0.2 μM) treatment for 6 hours for IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with MYC-EV or MYC-VANGL2 for 24 hours. ( F ) Co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with HA-K48–linked ubiquitin and MYC-EV or MYC-VANGL2; cell lysates were harvested after Baf A1 (0.2 μM) treatment for 6 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Luciferase, Activity Assay, shRNA, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Weight (A) and survival (B) of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice ( n = 6 mice per group) after intraperitoneal injection of VSV [1 × 10 8 plaque-forming units (PFU) per mouse]. ( C ) ELISA for IFN-β in serum of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice treated with phosphate-buffered saline (PBS) or infected with VSV (1 × 10 8 PFU per mouse) via intraperitoneal injection for 18 hours. ( D and E ) RT-PCR analysis of Ifnb (D) or VSV-G (E) mRNA in the spleen (left), lungs (center), and liver (right) from mice, as in (C). ( F ) Representative hematoxylin and eosin–stained images of lung sections from mice as in (C). Scale bars, 50 μm. ( G ) Graphical abstract to illustrate how VANGL2 negatively regulates IFN-I signaling upon virus infection. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Developmental biology

Article Title: Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

doi: 10.1016/j.ydbio.2015.10.022

Figure Lengend Snippet: Fig. 2. At E12.0 LVV-forming endothelial cells (LVV-ECs) delaminate from the veins in the luminal orientation. (A) Organization of veins and lymph sacs (LS) in the frontal orientation at this first step of LVV development. Lymphatic endothelial cells (LECs) and LVV-ECs that form LVVs are in yellow and red respectively. The remaining LECs that form LS and venous endothelial cells are in green and blue respectively. EJV is perpendicular to IJV and SVC. (B) Immunohistochemistry for the indicated markers in the region from panel A. LVV-ECs are indicated by arrows. PROX1 is expressed at higher level in LVV-ECs compared to LECs. FOXC2 and GATA2 are expressed almost exclusively in LVV-ECs. In contrast, PDPN is restricted to LECs. VEGFR3 is higher in LVV forming LECs compared to LVV-ECs and the rest of LECs. ITGA9 is strongly expressed in the LVV forming LECs and LVV-ECs. Arrowheads point to the blood cells seen within the lymph sacs. (C–E) After performing immunohistochemistry on sections as described above, the fluorescent signals produced by antibodies were measured in arbitrary units (a.u.) using ImageJ software. PROX1 (C), FOXC2 (D) and GATA2 (E) are expressed at significantly higher levels in LVV-ECs compared to LEC progenitors and LECs. (F) 800 μm sagittal section of an E12.0 ProxTom embryo was immunostained and imaged by confocal microscopy. Two loose clusters of Tomhigh LVV-ECs are seen within the vein. Dotted line represents the artery located between the LVV-ECs clusters. SEM of the same section revealed delaminating LVV-ECs that overlap each other (pseudo colored in magenta) in both anterior (white arrow) and posterior (yellow arrow) clusters. Statistics: n¼3 for B; n¼6 for F. For panels C–E, the indicated numbers of cells from a single embryo were analyzed. This data is representative of three-independent experiments. **** po0.0001. Abbreviations: LS, lymph sac; IJV, internal jugular vein; EJV, external jugular vein; SCV, subclavian vein; SVC, superior vena cava; A, artery. Scale bars: 50 μm for B and the top two panels of F ; 10 μm for the bottom two panels of F .

Article Snippet: We generated a new Foxc2þ / CreERT2 mouse line by replacing the open reading frame of Foxc2 with a cDNA for CreERT2 (Cyagen Biosciences, Santa Clara, CA, USA).

Techniques: Immunohistochemistry, Produced, Software, Confocal Microscopy

Journal: Developmental biology

Article Title: Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

doi: 10.1016/j.ydbio.2015.10.022

Figure Lengend Snippet: Fig. 7. Variable penetrance of edema phenotype in Foxc2þ/ embryos correlates with defective LVVs. Approximately half of the Foxc2-heterozygoes embryos generated in C57BL/6 background are grossly indistinguishable from wild type embryos (A, B). The other half develops edema (C, arrows) by E14.5. Analysis of frontal sections from these embryos revealed one LVV in the normal-looking Foxc2þ/ embryos (E, arrow). The edematous embryos lack LVVs (F, arrowheads). (G–I) SEM of the LVV-complex from control embryos revealed two LVVs (G, magenta) in control and one LVV (H, magenta) in non-edematous Foxc2-heterozygote embryos. In contrast, no LVVs are seen in edematous Foxc2-heterozygoes (I). Instead, a few elongated cells with the characteristics of LVV-ECs are seen (magenta). Statistics: n¼3 for A–G; n¼4 for H; n¼2 for I. Abbreviations: SVC, superior vena cava; SCV, subclavian vein; LS, lymph sac; IJV, internal jugular vein. Scale bars: 50 μm.

Article Snippet: We generated a new Foxc2þ / CreERT2 mouse line by replacing the open reading frame of Foxc2 with a cDNA for CreERT2 (Cyagen Biosciences, Santa Clara, CA, USA).

Techniques: Generated, Control

Journal: Developmental biology

Article Title: Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

doi: 10.1016/j.ydbio.2015.10.022

Figure Lengend Snippet: Fig. 9. GATA2 is necessary for the proper differentiation of LVV-ECs. (A–F) E13.5 Gata2 þ/ or TgVE;Gata2f/ (in which Gata2 is conditionally deleted from all endothelial cells using CreERT2 after tamoxifen injection at E10.0) embryos were analyzed using the indicated markers. A–D are 12 μm frontal cryosections. PROX1 (A, C arrows) and FOXC2 (C, arrows) are strongly expressed in LVV-ECs of Gata2þ/ embryos. In contrast, PROX1 (B, D arrows) and FOXC2 (D, arrows) are weakly expressed in the LVV-forming region of TgVE; Gata2f/ embryos. E and F are projections of 100 μm thick frontal cryosections imaged by confocal microscopy. LVVs (E, arrows) seen in Gata2þ/ are absent in TgVE; Gata2f/ embryos (F, arrows). Statistics: n¼3. Abbreviations: LS, lymph sac; IJV, internal jugular vein; SCV, subclavian vein; SVC, superior vena cava. Scale bars: 50 μm.

Article Snippet: We generated a new Foxc2þ / CreERT2 mouse line by replacing the open reading frame of Foxc2 with a cDNA for CreERT2 (Cyagen Biosciences, Santa Clara, CA, USA).

Techniques: Injection, Confocal Microscopy

Journal: Aging (Albany NY)

Article Title: Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

doi: 10.18632/aging.103450

Figure Lengend Snippet: Endothelin-1 induces senescence in mouse myoblasts (C 2 C 12 ) through ET A receptor. Cells were grown on coverslips ( A , C ) and incubated with 1 nM ET-1 at different times ( A , B ), or incubated with 10 μM Bosentan (Bos), 100 nM BQ-123 (BQ123) or 100 nM BQ-788 (BQ788) added 30 min before ET-1 (1 nM), and then incubated for 72h ( C ) or 48h ( D ). Then, senescence was tested measuring SA-ß-GAL activity (panel A , C ) and protein content from p16 (panel B , D ). Representative microphotographs are shown on the left with 40x magnification and the densitometric analysis is shown on the right panel A , C . Scale bar, 50 μm. A representative Western blot of p16 is shown next to the densitometric analysis on the panel B , D . Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells ( C or time 0), and **p<0.05 vs ET alone.

Article Snippet: C 2 C 12 , a mouse myoblast cell line, was purchase from American Type Culture Collection (Manassas, VA, USA).

Techniques: Incubation, Activity Assay, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

doi: 10.18632/aging.103450

Figure Lengend Snippet: Endothelin-1 increases FN expression in mouse myoblasts (C 2 C 12 ) through ET A receptor. Cells were incubated with 1 nM ET-1 at different times. Then, FN mRNA expression was assessed by RT-qPCR (panel A ) and FN protein content by Western blot (panel B ). To study the ET receptor implicated, cells were incubated with 100 nM BQ-123 (BQ123) or 100 nM BQ-788 (BQ788) added 30 min before ET-1 (1 nM), and then incubated for 24h. Then, FN protein content (panel C ) as well as intracellular FN (in red) and extracellular FN (in green) expression (panel D ) were studied by Western and immunofluorescence, respectively. In the experiments of the analysis of protein content, a representative Western blot is shown at the top with the densitometric analysis below (panel B, C ). Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells ( C ), and **p<0.05 vs ET alone.

Article Snippet: C 2 C 12 , a mouse myoblast cell line, was purchase from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Immunofluorescence, Control

Journal: Aging (Albany NY)

Article Title: Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

doi: 10.18632/aging.103450

Figure Lengend Snippet: Fibronectin induces senescence in mouse myoblasts (C 2 C 12 ) through integrin/ILK activation. Cells were grown on coverslips (panels A , C ) to test senescence measuring SA-ß-GAL activity and p16 protein content by Western blot (panels B , D ). ( A ) Cells were incubated with 2.5 μg/ml FN at different times to assess SA-ß-GAL activity by confocal microscopy. ( B ) Cells were incubated at different doses of FN for 48h to analyze p16 protein content. ( C ) Cells were incubated with 2.5 μg/ml FN or 1 nM ET-1 in the presence or not of 50 μM Tirofiban (TF) for 48h to assess senescence by SA-ß-GAL activity (panel C ) or by p16 protein content (panel D ). Representative microphotographs are shown at the top with 40x magnification and the densitometric analysis is shown below. Scale bar, 50 μm. A representative Western blot of p16 is shown at the top and the densitometric analysis is shown below. In panels C and D closed bars represent data of FN treatment and stripped bars represent data of ET-1 treatment; lane 1: control cells; lane 2: FN or ET alone; lane 3: FN or ET plus TF; lane 4: TF alone. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells ( C or time 0), and **p<0.05 vs ET or FN alone. ( E ) Cells were transfected with siRNA against ILK or scrambled as siControl to assess senescence by p16 protein content upon 2.5 μg/mL FN treatment for 48h. A representative Western blot of ILK and p16 are shown on the left panel and the densitometric analysis is shown on the right. Values are the mean±SEM of 3 independent experiments, *p<0.05 vs. control cells ( C from siControl).

Article Snippet: C 2 C 12 , a mouse myoblast cell line, was purchase from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activation Assay, Activity Assay, Western Blot, Incubation, Confocal Microscopy, Control, Transfection

Journal: Aging (Albany NY)

Article Title: Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

doi: 10.18632/aging.103450

Figure Lengend Snippet: Proposal mechanism of action of ET-1 on muscular fibrosis and senescence. Myoblast cells present both type of ET-1 receptors, ET A and ET B , which are inhibited by specific antagonists such as BQ123 and BQ788, respectively. The binding of ET-1 to ET A receptor induces fibrosis and senescence through ROS production by activation of PI 3 K-AKT-GSK pathway. The inflammation induced by ET-1 could be also implied. Several antagonists were used to inhibit both, ROS production with the antioxidant N-acetylcysteine (NAC), and the PI 3 K-AKT-GSK pathway with AKT inhibitor, LY-294,002 and Wortmannin, to check the mechanism. Fibronectin could induce senescence through integrin receptor activation by joining to RGD sequence, and then trigger some downstream pathways through ILK activation. Tirofiban blocks the joining of FN to RGD sequence. Findings in aged mice are showed below which are similar to those found in myoblast cells induced by ET-1, suggesting that the appearance of fibrosis and senescence could be involved in the genesis of sarcopenia related to aging. Note: all antagonists or inhibitors are represented in an orange box and unexplored mechanisms with an arrow with dashed pink line.

Article Snippet: C 2 C 12 , a mouse myoblast cell line, was purchase from American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Activation Assay, Sequencing