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: Aging (Albany NY)

Article Title: IL1- and TGFβ-Nox4 signaling, oxidative stress and DNA damage response are shared features of replicative, oncogene-induced, and drug-induced paracrine ‘Bystander senescence’

doi:

Figure Lengend Snippet: ( A ) Detection of cytokines in medium of parental senescent cells using FACS beads assay. The values are shown as a fold induction relative to non-treated BJ cells (ctrl). ( B ) Immunoblot detection of serine 139 phosphorylated H2AX (γH2AX) and STAT3 phosphorylated on tyrosine 705 (STAT3 pS705) in BJ cells treated with control (CM) or replicative senescent medium (RSM) in presence or absence of IL6 neutralizing antibody (2 μg/ml). ( C ) Immunoblot detection of γH2AX and STAT3 pS705 in BJ cells treated with CM or RSM in presence or absence of JAK kinase family specific inhibitor (JAKi; 0.25 μM). ( D ) Immunofluorescence detection of the p65 subunit of NFκB in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 4 days. BJ cells treated with medium from non-senescent BJ cells (CM) were used as a control. Bar 15μm. ( E ) Immunoblot and ( F ) indirect immunofluorescence detection of γH2AX in BJ cells treated with CM or RSM in presence or absence of IL1 receptor antagonist (IL1R ant.; 25 μM). Bar 15μm. ( G ) Immunoblot detection of γH2AX, NEMO and SMAD2 phosphorylated on serine 465/467 (SMAD2 pS465/467) and ( H ) immunofluorescence detection of γH2AX foci in BJ cells treated with CM or RSM in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM), NEMO siRNA or combination of both. Cells without transfection with siNEMO were transfected with control siRNA. Bar 15μm. ( I ) Detection of ROS production by 2',7'-dichlorofluorescein (DCF) staining in BJ cells treated with CM or RSM in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM). Bar 15μm. All these experiments (except experiment A and C ) were measured in BJ cells treated 4 days with conditioned medium from replicative senescent cells (RSM; diluted 1:1 with fresh medium). BJ cells treated 4 days with medium from non-senescent cells (CM; diluted 1:1 with fresh medium) were used as a control.

Article Snippet: JAK inhibitor I, TGF beta receptor 1 inhibitor II and IL1 receptor antagonist were purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Western Blot, Immunofluorescence, Transfection, Staining

Journal: Aging (Albany NY)

Article Title: IL1- and TGFβ-Nox4 signaling, oxidative stress and DNA damage response are shared features of replicative, oncogene-induced, and drug-induced paracrine ‘Bystander senescence’

doi:

Figure Lengend Snippet: ( A ) Nox4 mRNA levels quantified by real time qRT-PCR in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 20 days or ( B ) treated with recombinant TGFβ1 (1μM) for 4 days. BJ cells treated with medium from non-senescent BJ cells (CM) or non-treated cells (ctrl) were used as a control. The mRNA values represent average of two independent experiments and are shown as a fold induction relative to control BJ cells; error bars represent standard error. β-actin was used as a reference gene. ( C ) Detection of ROS production by 2',7'-dichlorofluorescein (DCF) staining in BJ cells in presence or absence of recombinant TGFβ, protein (4 days). ( D ) Nox4 mRNA levels quantified by real time qRT-PCR in BJ cells treated with replicative senescent cell medium (RSM) in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM) or IL1 receptor antagonist (IL1R ant.; 25 μM). The mRNA values represent average of two independent experiments and are shown as a fold induction relative to RSM BJ cells; error bars represent standard error. β-actin was used as a reference gene. ( E ) Detection of cytokines in medium of different bystander senescent cells 20 days after treatment using FACS beads assay. Fresh medium was added 24 hours before harvest to allow measurement of cytokines produced by bystander cells. The values are shown as a fold induction relative to BJ cells treated with medium from non-senescent BJ cells (CM). ( F ) Schematic representation of the IL1- and TGFβ-dependent induction of DNA damage and secondary (bystander) senescence common to three forms of primary (parental) senescence: senescence-associated secretome (SASP), especially IL1β and TGFβ, produced by three forms of senescent cells is able to induce DNA damage and bystander senescence in neighboring cells. Induction of secondary SASP in bystander cells indicates a possibility to spread DNA damage and senescence in surrounding tissue.

Article Snippet: JAK inhibitor I, TGF beta receptor 1 inhibitor II and IL1 receptor antagonist were purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Quantitative RT-PCR, Recombinant, Staining, Produced