Journal: Inflammation and Regeneration

Article Title: Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury

doi: 10.1186/s41232-024-00319-4

Figure Lengend Snippet: Early-stage tissue evaluation after neurite bundle-derived artificial nerve transplantation. a , b Immunohistochemistry images with anti-NFH and anti-CD31 antibodies. Dotted arrows indicate the proximal side of the intact nerve areas, and solid arrows indicate the NFH- and CD31-positive regeneration tip in the reconstructed tissue. Scale bars = 1 mm. c , d Quantitative comparison of axonal extension distance was indicated by NFH-positive areas and that of vascular elongation distance was demonstrated by CD31-positive areas at 1 and 2 weeks after transplantation. Significant differences between groups elongation distances were observed in 2-week samples only. e , f Iba1 immunohistochemistry image of the proximal part of the regenerated tissue at 2 weeks after transplantation (e). Scale bars = 1 mm. Quantitative analysis of Iba1-positive areas demonstrated that a larger number of macrophages aggregated in the Auto, motor TP and sensory TP (f). ( n = 3). * p < 0.05, ** p < 0.01, N.S. = not significant. Data are represented as the mean ± SEM

Article Snippet: The primary antibodies used in this study were human polyclonal anti-pan-ELAVL (ELAV-like protein 2/3/4) antibody (1:2000, kindly provided from Prof. Robert Darnell, Rockefeller University), rabbit polyclonal anti-TrkA antibody (1:500, kindly provided from Prof. Louis F. Reichardt, UCSF), chicken polyclonal anti-TrkB antibody (1:500, kindly provided from Prof. Louis F. Reichardt, UCSF), goat polyclonal anti-TrkC antibody (AF373, 1:200, R&D systems), rabbit polyclonal anti-parvalbumin antibody (PV-28, 1:1000, Swant), rabbit polyclonal anti-CGRP antibody (BML-CA1134, 1:500, Enzo Life Sciences), Goat polyclonal anti-Choline acetyltransferase antibody (AB144P, 1:200, Chemicon), Mouse monoclonal anti-Islet1 and Islet2 antibody (39.4D5, 1:200, DSHB), rabbit polyclonal anti-Neurofilament heavy polypeptide antibody (ab8135, 1:500, Abcam), goat polyclonal anti-mouse and anti-rat CD31 (AF3628, 1:100, R&D), rabbit polyclonal anti-Iba1 antibody (GtX100042, 1:500, GeneTex), goat anti-GFP antibody (600–101-215, 1:1000, Rockland).

Techniques: Derivative Assay, Transplantation Assay, Immunohistochemistry, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-φ neutralization abolishes ST-induced interferon production in zebrafish embryos. (A) Representative immunohistochemical staining of IFN-φ (brown DAB precipitate) in 1 dpf wild-type (WT) embryos under four conditions: (a.1) unstimulated control, (a.2) ST stimulation (10 6 cells/ml for 2h), (a.3) anti-IFN-β antibody pretreatment alone, and (a.4) anti-IFN-β pretreatment followed by ST stimulation. Scale bar: 50 μm. (B) Quantification of IFN-φ-positive cells from two slides per group (eight sections/slide, three embryos/section) using Fiji-ImageJ. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Neutralization, Immunohistochemical staining, Staining, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-I signaling is essential for IL-1β–mediated protection against ST stimulation. (A) Locomotor activity (total distance traveled) assessed 72h after stimulation at 28°C. Mortality count (C) and Caspy2 expression in WT embryos after ST stimulation (10 6 cells/ml, 2h) with or without anti-IFN-β antibody pretreatment. The corresponding bar graph (D) represents the protein expression levels as a percentage of the control. (E) Quantification and (F) representative IHC images of mature IL-1β (mIL-1β)–positive cells (brown DAB precipitate) in: (f.1) ST-stimulated embryos, (f.2) pretreatment with anti-IFN-β alone, and (f.3) pretreatment with anti-IFN-β followed by ST stimulation. Scale bar: 200 μm. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Effectiveness of CRISPR/Cas9 depletion of loc795232. Basal expression of loc795232 mRNA transcripts in unstimulated WT or KO embryos ( n = 100/group) at 1 dpf by RT-qPCR (A) or by in situ hybridization (B) . All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM of three biological replicates. Independent groups of 1 dpf embryos previously treated or not with Pam3CSK4 or MCC950 and subjected to ST stimulation for 2h, as well as KO larvae stimulated with ST were processed for Natterin detection by WB (C) using anti-Natterin serum (62 kDa dimeric form) and anti-IgG TrueBlot HRP. The corresponding bar graph represents the protein expression levels as a percentage of the control (D) , and Ponceau S staining is visualized in the first horizontal line.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: CRISPR, Expressing, Quantitative RT-PCR, In Situ Hybridization, Control, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is required for Gbp4 induction. Constitutive expression of gbp4 (A) and gbp1 (C) , was analyzed in unstimulated WT embryos ( n = 100/group) at 24h intervals by RT-qPCR. 1 dpf ST-responsive expression of gbp4 (B) and gbp1 (D) was assessed in WT and KO groups. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. * p < 0.05 versus unstimulated control WT; # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is essential for proteolytic activation of Caspy and Caspy2 during ST stimulation. (A) Developmental expression profile of caspy2 mRNA in unstimulated WT embryos ( n = 100/group) from 24 to 120 hpf. (B) One day post-fertilization ST-induced caspy2 expression in WT versus natterin knockout (KO) embryos 2h post-stimulation. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM; * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT. Western blot analysis of mature Caspy ( Supplementary Figure S3 ) and Caspy2 ( Supplementary Figure S4 ) expression in whole embryo lysates at indicated times post-ST stimulation (15 and 30 min, 1 and 2h). Data are normalized to the unstimulated control and presented as percentage values. Quantitative densitometry is shown in bar graphs (C, D) .

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activation Assay, Expressing, Knock-Out, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Neural plate stage Xenopus laevis embryos were processed for co-immunoprecipitation (IP) assays. Example of Western blot assay from immunoprecipitates with FOLR1 or GFP (control) antibodies and probed for CD2AP or FOLR1. Similar results were observed in N = 3 independent experiments. b Neural plate stage Xenopus laevis embryos were fixed and processed for immunostaining. Images are transverse single z-sections of immunostained neural plate showing apical colocalization of phospho-CD2AP (p-CD2AP) and p-c-Cbl with C-cadherin. Scale bar, 10 μm. Similar results were observed in N = 3 independent experiments. c Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 9.9 pmol of Control-morpholino (Control, Control-MO), 2.6 pmol CD2AP-MO1 (CD2AP KD1) or 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD2/KD) per blastomere until neural tube closed in control embryos, when they were fixed and photomicrographed. Examples of whole embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting closed (green) or open (purple, NTD) neural tube. Bar graph represents proportion of phenotypes in each group. d Two-cell stage Xenopus laevis embryos were unilaterally microinjected with 9.9 pmol Control-MO (Control) and 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD) along with GFP and mCherry mRNA, respectively, and allowed to develop until they reached mid-neural plate stages, when they were fixed and processed for immunostaining. Image is a transverse section of the neural plate, immunostained for GFP (Control), mCherry (CD2AP KD) and C-cadherin. Double arrows indicate apical surface length of medial superficial Control (white) and CD2AP KD (magenta) neural plate cells. Scale bar, 20 μm. Graph shows individual and mean ± SD apical length of superficial neural plate cells per embryo, n of cells = 75 in each half of the neural plate, N of embryos = 4. **** p < 0.0001, two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Immunoprecipitation, Western Blot, Control, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP and membrane mCherry mRNAs and unilaterally microinjected with 9.9 pmol CD2AP-MO (CD2AP KD) per blastomere along with fluorescent tracer and allowed to develop until they reached early neural plate stages (stage 13-14), when they were time-lapse imaged with an acquisition rate of 1 frame/6 min. Image is maximum intensity projection of single time frame. Dashed line indicates border between morpholino-injected and wild-type (WT) neural plate. Inset shows neural plate injected side showing tracer in blue. Graphs show distribution between both halves of the neural plate (in %) of the number of EEA1-GFP vesicles and area fraction of labeled endosomes per neural plate cell surface. Two-tailed paired t -test, n = 28 cells analyzed in each group from N = 5 embryos per group. Scale bar, 20 μm. b Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 7.4 pmol Control-MO (Control) or CD2AP-MO (CD2AP KD) per blastomere and allowed to grow until they reached mid-neural plate stages (stage 15–17) when neural plate was dissected and processed for Western blot assays. Image is an example of Western blot assay. Graph shows individual and mean ± SD percent of optical density (OD) for C-cadherin immunoblot band normalized with GAPDH protein band OD and compared to controls. Two-tailed ratio t -test, n = 28 and 24 neural plates for Control and CD2AP KD groups, respectively, N = 5 independent experiments. In ( a , b ), * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Membrane, Injection, Labeling, Two Tailed Test, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: Two-cell stage Xenopus laevis embryos were microinjected with 14.8 pmol Control-morpholino (MO, Control, a , b ), 3.2 pmol FOLR1-MO (FOLR1 KD, a ) or 14.8 pmol CD2AP-MO2 (CD2AP KD, b ) per embryo and incubated with saline or proteasome and lysosome inhibitors at the end of gastrulation (stage 12) until neural plate stages (stage 17) when they were processed for Western blot assays. Images are examples of Western blot assays. Graphs show individual and mean ± SD percent of optical density (OD) for CD2AP ( a ) or FOLR1 ( b ) immunoblot band normalized with GAPDH protein band OD and compared to controls. In ( a ), n = 34 and 42 neural plates for Control and FOLR1 KD, respectively, N = 7 independent experiments; n = 20 and 26 neural plates for Control+inhibitors and FOLR1 KD+inhibitors groups, respectively, N = 3 independent experiments. In ( b ) n = 16 and 20 for Control and CD2AP KD groups, respectively and n = 16 and 18 neural plates for Control+inhibitors and CD2AP KD+inhibitors, respectively, N = 3 independent experiments. In ( a , b ) * p < 0.05, *** p < 0.001, ns: not significant, two-tailed ratio t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Control, Incubation, Saline, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a – c Neural plate from mid neural plate stage Xenopus laevis embryos was dissected and dissociated cells plated in vitro. After 2 h, cells were loaded with the Ca 2+ sensor Fluo4-AM and time-lapse imaged. a Example of 1-h recording of neural plate cell Ca 2+ activity. b , c Folinic acid ( b , c ), folic acid ( c ) or vehicle was added to neural plate cells in culture during time-lapse imaging and the Ca 2+ response was recorded in the first minute post addition. b Example of acute transient elicited by 100 μM folinic acid. c Graph shows mean ± SEM folinic- or folic acid-responsive neural plate cells compared to total number of cells with spontaneous Ca 2+ transients in 30 min recording, N = 3 independent experiments. Two-tailed one sample t and Wilcoxon test. d Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA and grown until early and mid-neural plate stages when they were time-lapse imaged before and after addition of vehicle or 300 μM folinic acid. Image shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles. Graph shows individual and mean ± SD percent change in Ca 2+ transient frequency before and after addition of vehicle or folinic acid, n = 4, 5, 6 and 7 embryos for Early-Vehicle, Early-Folinic acid, Mid-Vehicle and Mid-Folinic acid groups, respectively. One-sample two-tailed t -test, compared to hypothetical value of 100%. e – h Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA ( e – h ) and unilaterally microinjected with 9.9 pmol FOLR1-MO1 (FOLR1 KD1/KD) or 1.6 pmol FOLR1-MO2 (FOLR1 KD2) per blastomere ( e ) and grown until mid-neural plate stages when they were time-lapse imaged. Image in ( e ) shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles in WT and FOLR1 KD1 halves. Graphs show individual Ca 2+ transient frequency (transients/5 min) in WT and FOLR1 KD1 or KD2 halves ( e , n = 6 embryos) and in WT embryos before and after addition of vehicle ( f , n = 7 embryos), 50 μM folic acid ( g , n = 6 embryos), Na + and voltage-gated Ca 2+ channel blockers (VGC block : 0.02% tricaine+10 μM nitrendipine+25 μM TTA-2, h , n = 5 embryos), or a mix of folic acid and VGC block ( h , n = 5 embryos). Two-tailed paired t -test ( e – g ) and 1-way ANOVA-Tukey multiple comparisons test ( h ). In ( c – h ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. i Model of FOLR1 and CD2AP regulation of neural tube formation. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: In Vitro, Activity Assay, Imaging, Two Tailed Test, Blocking Assay