Journal: Frontiers in Molecular Biosciences

Article Title: Sequence- and structure-specific RNA oligonucleotide binding attenuates heterogeneous nuclear ribonucleoprotein A1 dysfunction

doi: 10.3389/fmolb.2023.1178439

Figure Lengend Snippet:

Article Snippet: Additionally, for this study we mutated amino acid R55A in OptoA1 using HiFi DNA assembly cloning (NEBuilder HiFi DNA Assembly Cloning Kit, New England BioLabs) to generate N-pmCry2PHR-A1 (R55A)-mCherry-C.

Techniques: Emulsion, Lysis, Protease Inhibitor, Protein Extraction, Modification, Membrane, Staining, Western Blot, Clone Assay, Recombinant, Software, Microscopy

Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: Translating RMC-6236 activity in NSCLC. A, Efficacy of RMC-6236 on Kras G12C , Kras G12D , Kras G12V , Kras G12A , Kras G13D , or Kras Q61H -driven autochthonous lung tumors in immunocompetent mice. A pool of lentiviral cDNA vectors encoding each oncogenic Kras variant was delivered intratracheally to the lungs of each mouse, and 13 weeks after tumor growth, mice were treated with RMC-6236 at 20 mg/kg po qd for 3 weeks prior to analysis. 95% confidence intervals are shown. B, Efficacy of RMC-6236 and adagrasib in the LUN055 NSCLC PDX model with KRAS G12C allele copy-number gain. Immunoblot Western analyses (left) of RAS and KRAS protein levels in NCI-H358 ( KRAS G12C/WT , NSCLC), LU99 ( KRAS G12C/WT , NSCLC), NCI-H2122 ( KRAS G12C/G12C , NSCLC), and LUN055 ( KRAS G12C/WT , NSCLC) xenograft tumors. Relative copy-number (middle) of KRAS WT or KRAS G12C in LUN055 xenograft tumors ( n = 2) were determined by ddPCR and normalized to ACTB . LUN055 xenograft tumor-bearing mice were treated with vehicle or RMC-6236 at 25 mg/kg po qd or adagrasib at 100 mg/kg po qd for 24 to 28 days ( n = 3 per group, right). Mean tumor volumes of each group were plotted over the course of treatment. Dotted line indicates the initial average tumor volume. Error bars, SEM. C, Efficacy of RMC-6236 in the intracranially implanted LU99-Luc ( KRAS G12C/WT , NSCLC) xenograft model ( n = 8 per group). RMC-6236 was dosed at 25 mg/kg daily for 21 days. Images of bioluminescence in individual mice were shown. Bioluminescence of ROI in vehicle control and RMC-6236 groups were compared by two-way repeated-measures ANOVA at day 21 (**, P < 0.01). Results were shown as mean ± SEM. D, Antitumor activity of RMC-6236 and the combination with anti–PD-1 (clone RMP1-14, rat IgG2a) following repeated administration in BALB/c mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 and anti–PD-1 treatment started on day 17 after implantation. RMC-6236 treatment was stopped at day 31 after implantation and anti–PD-1 at day 35 after implantation. E, Antitumor activity of RMC-6236 following repeated administration in NSG mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 treatment started on day 16 after implantation. F, Immune cell composition (CD8 + and CD4 + T cells, Ly6C + and Ly6G + myeloid-derived suppressor cells and M2 macrophages) in murine colon carcinoma eCT26 syngeneic tumors ( Kras G12C/G12C ) represented as percentage of CD45 + cells and expression of cell-surface markers on viable, CD45 − large cells (assessed as tumor cells) 24 hours post 4 days of treatment with vehicle or RMC-6236 at 25 mg/kg po qd n = 3 biological replicates/group represented as mean; *, P < 0.05; **, P < 0.01; ns, nonsignificant by two-sided Student t test.

Article Snippet: Probes and primers for the following genes were included in the multiplexed ddPCR: KRAS WT [dPCR Mutation Detection Assay KRAS Wild-Type for p.G12C, Human (apexbio, AA100902-WT)], KRAS G12C [dPCR Mutation Detection Assay KRAS Mutant for p.G12C, Human (apexbio, AA100902-MU)], and ACTB ( ACTB probe, 5′-Cy5-ATTGCCGACAGGATGCAGAAGGA-3′; ACTB primer F, 5′-GACATCCGCAAAGACCTGTA-3′; ACTB primer R, 5′-GGAAAGACACCCACCTTGAT-3′).

Techniques: Activity Assay, Variant Assay, Western Blot, Control, Injection, Derivative Assay, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 1. Tumor-associated soluble uPAR (s-uPAR) enhances HUVEC invasion, migration and angiogenesis. (a) Conditioned medium (CM) was collected from tumor cells (parental and stably expressing empty vector (EV), uPAR-cDNA (UR) and uPAR siRNA (UR-Si)). Immunoblot analyses were performed for s-uPAR and DDK using specific antibodies. (b) s-uPAR levels in CM were quantified using uPAR Quantikine Immunoassay kit. Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental control. (c) Cells were labeled (tumor cells: Qtracker-525-Green and HUVECs: Qtracker- 655-Red) and seeded into separate chambers of culture inserts. After 16 h, the culture inserts were removed and cells were allowed to migrate for a further 24 h. Images were captured at 0 and 24 h of incubation and cell migration was quantified using ImageJ software (NIH). The levels of HUVEC migration were normalized to HUVEC migration in parental cells and are represented as arbitrary units. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental control. (d) HUVEC invasion experiments were performed using ThinCertTM inserts as described in Materials and methods. The levels of HUVEC invasion was quantified and normalized to HUVEC invasion in parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (e, f) In vitro angiogenesis assay was performed as described in Materials and methods. The degree of angiogenic induction by CM was quantified by ImageJ software (NIH) for the numerical value of the product of the relative capillary length per microscopic field. Serum-free medium (SFM) and recombinant human uPAR (rh-uPAR) in SFM were used as controls (insets). Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM. uPAR antibody, uPAR-Ab; isotype control, NSp.IgG. (g) Migration assay was performed using CM. In this case, both chambers of culture inserts were seeded with HUVECs. After 16 h, the culture inserts were removed, CM was added and cells were allowed to migrate for 24 h. Invasion assay was performed as described above. uPAR-Ab. or Nsp.IgG were added to UR-CM before adding onto cells. rh-uPAR was added to SFM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Labeling, Incubation, Software, In Vitro, Angiogenesis Assay, Recombinant, Invasion Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 2. s-uPAR recruits onto HUVEC membrane. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured on CM for 24 h, labeled with anti-uPAR antibody, followed by Alexa Fluor-488-conjugated secondary antibody and were analyzed by fluorescence-activated cell sorting (FACS) for uPAR expression. Serum-free medium (SFM) and rh-uPAR were used as controls. Isotype control (Neg.). (b) HUVECs were cultured in chamber slides on CM for 24 h and fixed in 4% paraformaldehyde and 0.2% glutaraldedyde in phosphate-buffered saline for 1 h. Immunocytochemical analysis was performed as described in Materials and methods. Isotype control (Neg.; inset). Slides were mounted and photographed. (c) Equal amounts of proteins were used for the extraction of HUVEC membrane fractions and were subjected to immunoblot analysis for uPAR expression using specific antibodies. The blot was re-probed for DDK-tag expression.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Membrane, Cell Culture, Labeling, FACS, Expressing, Control, Saline, Extraction, Western Blot

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 3. s-uPAR colocalizes in lipid rafts on HUVECs. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured in chamber slides on CM for 24 h and incubated with anti-uPAR antibody followed by Alexa Fluor-488- conjugated secondary antibody at 4 1C. Cells were again labeled with Alexa Fluor-595-CTxB subunit. Slides were mounted and analyzed by confocal microscopy. Negative controls, using an isotype antibody, showed no staining (inset). Serum-free medium (SFM) and DDK-tag containing rh-uPAR were used as controls. To disrupt lipid rafts, HUVECs were pretreated with MBCD, as described in Materials and methods. (b) HUVECs lipid rafts were isolated as described in Materials and methods. Lipid raft-enriched fractions were analyzed for uPAR and DDK-tag levels using immunoblot analysis. Flotillin-1 and caveolin-1 served as controls. Protein band intensities were quantified by densitometric analysis using ImageJ software (NIH). The levels of uPAR protein were normalized to protein levels in HUVECs cultured on parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (c) Invasion and migration assays were performed as described in Figure 1d In vitro angiogenesis assay was performed as described in Figure 1. To deplete cholesterol, HUVECs were pretreated with MBCD as described in Materials and methods (c and d). Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Incubation, Labeling, Confocal Microscopy, Staining, Isolation, Western Blot, Software, Migration, In Vitro, Angiogenesis Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 4. s-uPAR induces ERK/Rac1-mediated migration and tube formation in HUVECs. Conditioned medium (CM) was collected from tumor cells, as described in Materials and methods. (a) HUVECs lysates were used to perform GST-Rac1 pull-down assay. The protein complexes were subjected to immunoblot analysis to detect active Rac1. Rac1 from total cell lysates was used as a control. (b) Total cell lysates were subjected to immunoblot analysis for phospho-ERK1/2 (pERK1/2) and total ERK1/2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. HUVECs grown on rh-uPAR were used as a control. (c) HUVECs were cultured on CM alone and/or supplemented with functional blocking anti-uPAR antibody (uPAR-Ab) or isotype control (Nsp.IgG.) or MEK inhibitor (U0126) for 24 h. Cell lysates or GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1, Rac1 pERK1/2 and ERK1/2. GAPDH served as a loading control. (d) HUVECs were transfected with dominant-negative mutant Rac1 (Dn-Rac1) for 24 h and cultured on UR-CM. Micrographs were captured for green fluorescent protein (GFP) expression (green) and phase contrast (gray) immediately after the addition of UR-CM (magnification 60). (e) HUVECs were transfected with Dn-Rac1 for 24 h, cultured on CM for another 24 h, collected and lysed. GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1. GFP and Rac1 from total cell lysates were used as controls. (f) HUVECs were transfected with Dn-Rac1 for 24 h and cultured on CM alone and/or supplemented with uPAR-Ab., or Nsp.IgG or U0126 for another 24 h. Invasion and migration assays were performed as described in Figure 1. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **Po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Pull Down Assay, Western Blot, Control, Cell Culture, Functional Assay, Blocking Assay, Transfection, Dominant Negative Mutation, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 5. Diverse forms of tumor-associated s-uPAR in vitro and in vivo. (a) Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. CM was subjected to deglycosylation using a deglycosylation kit and analyzed by immunoblot for uPAR using specific antibodies. (b) Equal amount of proteins containing HUVEC lysates were used for extraction of cell membrane fractions and were subjected to deglycosylation, and analyzed by immunoblot for uPAR using specific antibodies. (c) In vivo angiogenic assay was performed by using the dorsal air sac model. 4910EV (EV), 4910UR (UR), 4910UR-Si (UR-Si) cells or a recombinant human uPAR (rh-uPAR) containing chamber was implanted in the dorsal cavity of mice. The micrographs for the presence of tumor-induced neovasculature (microvessels with curved thin structures and many tiny bleeding spots) and pre-existing vasculature (straight) were captured. Representative micrographs are shown. (d, e) Blood was collected from mice orthotopically xenografted with stably expressing EV, UR and UR-Si cells. Total uPAR levels were estimated using a commercial human uPAR Quantikine Immunoassay kit according to the manufacturer’s instructions. The data quantification for a set I (n ¼ 4; d) and set II (n ¼ 6; e), on day 15 and 40, respectively, after cell implantation are shown. Columns: mean; bars: s.d.; *Po0.01 vs parental control. (f) Blood serum (from mice 1–6; on day 40) was subjected to deglycosylation and analyzed by immunoblot for uPAR using specific antibodies. D2-D3, D2-D3 domain containing truncated s-uPAR; D3, D3 domain containing truncated s-uPAR; FL, full-length s-uPAR; .

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Extraction, Membrane, Recombinant, Stable Transfection, Expressing, Control

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 6. uPAR overexpression enhances tumor growth, vascularity and s-uPAR recruits onto endothelial cells in vivo. (a) Stably expressing EV, UR and UR-Si cells were injected intracerebrally into mice. Mice were euthanized and brains were collected and fixed as described in Materials and methods. Brain sections were stained with hematoxylin and eosin (H&E) solution, and representative micrographs are shown (upper panel). H&E-stained micrographs showing the tumor invasive front ( 20; lower panel). (b) Brain tumor areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Rockville, MD, USA). Columns: mean; bars: s.d.; n ¼ 6; *Po0.01 vs parental controls. (c) Immunohistochemical analysis of brain sections using anti-uPAR and anti-vascular endothelial growth factor (VEGF). Blood vessels in tumor sections were visualized with biotin-labeled tomato lectin. Inset: isotype control. (d, e) Fluorescence microscopy for colocalization of an endothelial cell marker (von Willebrand factor (vWF)/anti-CD31) and DDK-tag in tumor sections from mice that were implanted with 4910 EV (EV) and 4910UR (UR) cells. Inset, isotype control.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Staining, Software, Immunohistochemical staining, Labeling, Control, Fluorescence, Microscopy, Marker

Journal: PLoS Genetics

Article Title: Identification of the First ATRIP–Deficient Patient and Novel Mutations in ATR Define a Clinical Spectrum for ATR–ATRIP Seckel Syndrome

doi: 10.1371/journal.pgen.1002945

Figure Lengend Snippet: A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm −2 UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm −2 ) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.

Article Snippet: Anti-FANCD2, ATRIP and phospho-Chk1 (Ser317) antibodies were from Novus (Littleton), Bethyl (Montgomery), and Cell Signaling (Beverly, Woburn), respectively.

Techniques: Immunofluorescence, Western Blot, Expressing, Control, Comparison

Journal: PLoS Genetics

Article Title: Identification of the First ATRIP–Deficient Patient and Novel Mutations in ATR Define a Clinical Spectrum for ATR–ATRIP Seckel Syndrome

doi: 10.1371/journal.pgen.1002945

Figure Lengend Snippet: A) Analysis of the G2/M checkpoint defect in CV1720 cells following expression of ATRIP cDNA. G2/M checkpoint arrest was examined 2 h post exposure to 5 Jm −2 UV. As shown in , WT cells showed proficient checkpoint arrest whilst DK0064 (ATR–SS) and CV1720 (patient) cells are unable to undergo arrest. Expression of WT ATRIP cDNA restored the ability of CV1720 (patient) and DK0064 (ATR–SS) to undergo checkpoint arrest but this was not observed following transfection of cDNA encoding R760* ATRIP. Significantly, expression of ATRIP R760* did not impair checkpoint arrest in WT cells verifying that it does not exert a dominant negative impact. represent the mean and SD of three experiments. WT cells were GM2188. ATR–SS represents DK0064 and patient, CV1720. B) R760* ATRIP impairs ATR–ATRIP interaction. Crude lysates were prepared from HEK293T cells and either mock transfected (lane1), transfected with HA-tagged WT ATRIP cDNA (lane2), or R760* ATRIP cDNA (lane3) (generating p.Arg760* ATRIP protein) together with ATR cDNA. The extracts were immunoprecipitated with agarose-conjugated rabbit anti-HA-tag antibody (MBL). Interaction with ATR was examined by immunoblotting with ATR antibodies (left panel). Immunoblotting using the HA-tag (ATRIP; right panel) verified expression of the appropriately sized ATRIP in the samples. 33% of the crude lysate was loaded; IP, immunoprecipitate.

Article Snippet: Anti-FANCD2, ATRIP and phospho-Chk1 (Ser317) antibodies were from Novus (Littleton), Bethyl (Montgomery), and Cell Signaling (Beverly, Woburn), respectively.

Techniques: Expressing, Transfection, Dominant Negative Mutation, Immunoprecipitation, Western Blot

Journal: PLoS Genetics

Article Title: Identification of the First ATRIP–Deficient Patient and Novel Mutations in ATR Define a Clinical Spectrum for ATR–ATRIP Seckel Syndrome

doi: 10.1371/journal.pgen.1002945

Figure Lengend Snippet: A) Photographs of patient included with informed consent of parent. B) Cell extracts (50 µg) from LBLs derived from WT (IM257), patient 27-4BI or patient 19-8BI were immunoblotted using the indicated antibodies. Reduced expression of ATR was observed in both patients. 27-4BI also had reduced ATRIP expression. C) Structure of ATR showing the site of the mutations identified and the UME domain. D) The UME domain is conserved between species and the methionine residue within this domain is conserved in yeast.

Article Snippet: Anti-FANCD2, ATRIP and phospho-Chk1 (Ser317) antibodies were from Novus (Littleton), Bethyl (Montgomery), and Cell Signaling (Beverly, Woburn), respectively.

Techniques: Derivative Assay, Expressing, Residue

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b , e , f Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( a , b ) or EPC ( e , f ) cells. The cells were collected at 36 h ( a , e ) or 48 h ( b , f ) post-transfection. c , d , g , h Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( c , d ) or EPC ( g , h ) cells for 24 h, followed by infection with GCRV for another 24 h ( c , g ) or 36 h ( d , h ). i – l Expression of irf3 , irf7 , and tbk1 mRNA after bmp8a knockdown in ZFL cells. The cells were collected at 36 h ( i ) and 48 h ( j ) post-knockdown or at 24 h ( k ) and 36 h ( l ) post-infected with GCRV. m , o Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( m ) or EPC ( o ) cells. The cells were collected at 36 or 48 h post-transfection for Immunoblot analysis. n , p Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( n ) or EPC ( p ) cells for 24 h, followed by infection with GCRV for another 24 or 36 h. q , r Immunoblot analysis of phosphorylated (p-) TBK1 and IRF3 after bmp8a knockdown in ZFL cells. The cells were collected at 36 and 48 h post-knockdown or at 24 and 36 h post-infected with GCRV. s – u EPC cells were cotransfected with IFN-φ1pro-luc (200 ng, s ), IFN-φ3pro-luc (200 ng, t ) or EPC IFNpro-luc (200 ng, u ), and bmp8a (100 ng) together with each of the dominant negative plasmids including tbk1–K38M (100 ng), irf3DN (100 ng) and irf7DN (100 ng). At 48 h post-transfection, the cells were collected for luciferase assays. Renilla luciferase was used as the internal control. v – y Expression of irf3 , irf7 , and tbk1 mRNA in the liver, kidney, intestine, and spleen from WT or bmp8a −/− zebrafish injected i.p. with 50 µl of GCRV (10 8 TCID 50 per ml). The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell post hoc tests for comparison of multiple groups. All data were presented as mean ± SD (** p < 0.01, *** p < 0.001).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Western Blot, Dominant Negative Mutation, Luciferase, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b Expression of alk2 , alk3 , alk6a , bmpr2a , bmpr2b , actr2a , and actr2b mRNA in ZFL cells stimulated with poly(I:C) (2 μg/ml, a ) or GCRV (5 × 10 4 TCID 50 per ml, b ) for 48 h. c – e Expression of ifnφ1 ( c ) and ifnφ3 ( d ) mRNA in ZFL cells or EPC ifn ( e ) in EPC cells which were transfected with 2 μg of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, actr2b or empty vector for 48 h. f , h Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of pcDNA3.1-alk6a or empty vector in ZFL ( f ) or EPC ( h ) cells for 48 h. g , i Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of of pcDNA3.1-alk6a or empty vector in ZFL ( g ) or EPC ( i ) cells for 24 h, followed by infection with GCRV for another 36 h. j Schematic drawing of the alk6a-ΔGS mutation that the GS domain of Alk6a was deleted. k , m Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( k ) or EPC ( m ) cells for 48 h. l , n Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( l ) or EPC ( n ) cells for 24 h, followed by infection with GCRV for another 36 h. The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell posthoc tests for comparison of multiple groups. All data were presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, ns means no significant difference).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: Upon virus infection, the transcriptions of bmp8a are activated through the Jak-Stat1 pathway. The Bmp8a binds to BMP type I receptor Alk6a, promoting phosphorylation of Tbk1 and Irf3 to induce the expression of Ifn through p38 MAPK pathway.

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Infection, Expressing