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: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a – d Relative mRNA expression of NTF3 , ASCL1 , MYOD1 , and IL1RN in HEK293T cells transfected with dCas9-VP64 or dCas9-VP64-FUS and a single guide RNA (gRNA) targeting each gene promoter as well as a scrambled gRNA (gScr) control. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. e , f Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells transfected with dCas9-VPR or dCas9-VPR-FUS and a gRNA targeting each gene promoter. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. g , h Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells transfected with dCpf1-VP64 or dCpf1-VP64-FUS and a gRNA targeting each gene promoter. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a Boxplot illustrating the GFP intensity in HEK293R cells expressing different dCas9 activators together with gTetO. The results are presented as the median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 17198, 17379, 9902). b , c Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells expressing different dCas9 activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64-FUS group. d , e Fluorescence recovery after photobleaching (FRAP) analysis of HEK293R cells expressing BFP-tagged dCas9-VP64-FUS or dCas9-VP64-TDP-43 with gTetO. Up, representative timelapse images after photobleaching. Yellow arrowheads indicate bleached condensates. Scale bar, 10 μm. Down: FRAP curves showing mean ± SD fluorescence recovery of condensates ( n = 5 puncta per group). f Boxplot illustrating the GFP intensity in HEK293R cells expressing different dCas9 activators together with gTetO. The results are presented as the median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 19,096, 16,734, 13,743, 14,080, 12,071, 17,691). g , h Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells expressing different dCas9 activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64-FUS group. i Co-immunoprecipitation (co-IP) of Flag-tagged dCas9 activators and BRG1, MED1 or RPB1. Three independent experiments were performed and similar results were obtained. j , k Enrichment of Flag-tagged dCas9 activators, BRG1, RPB1, and MED1 at the NTF3 or IL1RN promoter. Data are presented as mean values ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Fluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a , b Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with a single gRNA targeting the indicated genes along with the labeled dCas9-activators. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64 or the dCas9-VP64-FUS group. c , d Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with dCas9-VP64-FUS-Shank3, dCas9-VP64-FUS-Shank3MA, or dCas9-VP64-FUS-Shank3 ME and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA test versus the dCas9-VP64-FUS-Shank3 group. e , f Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with dCas9-VP64-FUS-HOTag activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA test versus the dCas9-VP64-FUS group. g Relative mRNA expression of NTF3 , ASCL1 , MYOD1, and IL1RN in HEK293T cells transfected with a single gRNA targeting the indicated genes along with the dCas9-VP64-FUS-HOTag3 or dCas9-VP64-HOTag3-FUS. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. h Boxplot showing relative GFP intensity in 1xTetO-GFP, 7xTetO-GFP or 14xTetO-GFP reporter cells expressing dCas9, dCas9-VP64, dCas9-VP64-FUS, dCas9-VP64-FUS-Shank3 or dCas9-VP64-FUS-HOTag3 together with gTetO. The results are presented as the relative median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 24,461, 17,298, 21,807, 17,611, 11,280, 22,233, 14,347, 16,136, 15,605, 13,372, 23,028, 11,938, 15,760, 15,641, 12,923). Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Transfection, Labeling

Journal:

Article Title: Defining therapeutic targets by using adenovirus: Blocking NF-?B inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators

doi:

Figure Lengend Snippet: Inhibition of NF-κB through AdvIκBα infection has only marginal effects on the spontaneous production of anti-inflammatory mediators. Shown is the effect of infection of rheumatoid synovial cells with 40:1 of either Adv0 or AdvIκBα on the spontaneous production of IL-10 (A), the IL-1 receptor antagonist (B), the p75 soluble TNF receptor (C), and IL-11 (D). Error bars indicate SEM (n = 6).

Article Snippet: After incubation with adenovirus for 2 h, one well from each category was untreated, one was treated with 20 μg/ml of human recombinant IL-1 receptor antagonist (Cambridge Bioscience, Cambridge, U.K.), one was treated with 20 μg/ml of the A2 anti-TNFα antibody (Centocor), and one was treated with both of these cytokine inhibitors.

Techniques: Inhibition, Infection

Journal:

Article Title: Defining therapeutic targets by using adenovirus: Blocking NF-?B inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators

doi:

Figure Lengend Snippet: Effect of AdvIκBα infection on MMP and TIMP production in rheumatoid synovial cell cultures in which IL-1 and/or TNFα were neutralized. Rheumatoid synovial cells were either left uninfected or infected with 40:1 of Adv0 or AdvIκBα. After the virus was taken off and culture medium was added, the cells in each of the three categories either were left untreated or were treated with 20 μg/ml of the IL-1 receptor antagonist and/or 20 μg/ml of the A2 anti-TNFα antibody. The spontaneous production of MMP 1 (A), MMP 3 (B), and TIMP-1 (C) was assayed after 2 days. Shown is a representative of three complete experiments.

Article Snippet: After incubation with adenovirus for 2 h, one well from each category was untreated, one was treated with 20 μg/ml of human recombinant IL-1 receptor antagonist (Cambridge Bioscience, Cambridge, U.K.), one was treated with 20 μg/ml of the A2 anti-TNFα antibody (Centocor), and one was treated with both of these cytokine inhibitors.

Techniques: Infection