Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Lynx1 deletion increases nAChR sensitivity. (A) Example traces of spontaneous miniature endplate potential (MEPP) recordings from control and Lynx1 −/− extensor digitorum longus (EDL). (B) The average MEPP amplitude of control and Lynx1 −/− muscle, where the line width represents the SEM of 100 recordings. (C) The average rise time to peak amplitude of MEPPs represented in (B) . (D) The mean amplitude of MEPPs in control and Lynx1 −/− muscle. (E) The frequency of MEPPs in control and Lynx1 −/− muscle. (F) The average slope of MEPPs to peak amplitude in (B) . Control n ≥ 5, Lynx1 −/− n ≥ 8. All values are mean ± SD. * p < 0.05, unpaired, two-tailed Student’s t -test.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Lynx1 levels track structural and functional changes at neuromuscular junctions (NMJs). (A,B) qPCR analysis of Lynx1 mRNA levels in (A) developing tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and in (B) C2C12 myotubes at 3 and 7 days post-fusion compared to unfused myoblasts; * p < 0.05 versus P1 or control. † p < 0.05 versus 3 days post-fusion, one-way ANOVA with Bonferroni post hoc . (C,D) Representative images of Lynx1 (green) and fluorescently conjugated α-bungarotoxin (fBTX)-labeled nicotinic acetylcholine receptors (magenta) in control and Lynx1 −/− TA muscle cross-sections. (E,F) qPCR analysis of Lynx1 mRNA levels in (E) the TA muscle following fibular nerve crush injury, (F) C2C12 myotubes following 24-h carbachol (CCH) treatment, (G) the TA muscle of vesicular acetylcholine transporter (VAChT) knockdown (KD) mice, and (H) C2C12 myotubes following 24-h z-agrin treatment. * p < 0.05 versus P1 or control, one-way ANOVA with Bonferroni post hoc . All values are mean ± SD. Scale bar = 25 µm.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Functional Assay, Muscles, Control, Labeling, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Lynx1 reduces synaptic plasticity. (A) Example recordings of endplate potentials (EPPs) elicited by paired-pulse stimulation (0.2 Hz, 10-m interval) from control and Lynx1 −/− extensor digitorum longus (EDL). (B) The average EPP amplitude (measured of the first EPP of the pair) and (C) the average quantal content following paired-pulse stimulation. (D) The amplitude of EPPs at baseline and following tetanic stimulation (120 Hz, 10 s). The orange arrow denotes rapid depolarization following initial stimulation. The green arrow denotes post-tetanic potentiation in Lynx1 −/− but not control muscle. The blue arrow denotes the absence of long-lasting depression in Lynx1 −/− muscle. (E) Neuromuscular fatigue represented as relative strength, as a percent of baseline, following super-imposed muscle stimulations after fatigue protocol in 4-month-old control and Lynx1 −/− EDL (red dotted line represents SEM). Values in (B) and (C) are mean ± SD, and values in (D) and (E) are mean ± SEM. * p < 0.05, unpaired, two-tailed Student’s t -test.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Loss of Lynx1 has no discernable impact on NMJ development. (A–D) Representative images of NMJs in the extensor digitorum longus (EDL) muscles of P9 (A,C) and P21 (B,D) control and Lynx1 −/− mice. Motor axons were labeled with YFP (green) and nicotinic acetylcholine receptors (nAChRs) were labeled with fluorescently conjugated α-bungarotoxin (fBTX, magenta). (E–H) Morphological analysis of neuromuscular junctions (NMJs), including (E) the degree of NMJ innervation, (F) the percentage of NMJs with axonal sprouts, (G) the percentage of NMJs innervated by more than one axon, and (H) NMJ area, as determined by the area of nAChR clusters. All values are mean ± SD. † Age effect, p < 0.05, two-way ANOVA. * p < 0.05 versus age-matched control, two-way ANOVA with Šídák’s multiple comparisons test. n ≥ 3. Scale bar = 20 µm.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Muscles, Control, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Lynx1 deletion increases the incidence of age-related changes at adult NMJs. (A,B) Representative images of neuromuscular junctions (NMJs) in the extensor digitorum longus (EDL) of 7-month-old (A,C) and 12-month-old (B,D) control and Lynx1 −/− mice. Motor axons were labeled with YFP (green), and nicotinic acetylcholine receptors (nAChRs) were labeled with fluorescently conjugated α-bungarotoxin (fBTX, magenta). (E) The percentage of innervated NMJs, either fully, partially, or not at all. (F–M) Morphological analysis of NMJs, including (F) the presence of axon sprouts, (G) multiple innervations, (H) the presence of preterminal axon blebs, (I) the presence of blebs in the axon terminal, (J) nAChR area, (K) endplate area, (L) nAChR cluster dispersion, and (M) nAChR fragmentation. n = 3–4. * p < 0.05 versus age-matched control; † age effect, p < 0.05; ‡ genotype effect, p < 0.05; two-way ANOVA with Šídák’s multiple comparisons test. All values are mean ± SD. Scale bar = 20 µm.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Control, Labeling, Dispersion

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice

doi: 10.3389/fcell.2022.838612

Figure Lengend Snippet: Loss of Lynx1 does not affect the stability nor repair of neuromuscular junctions (NMJs) following denervation. (A,B) Representative images of NMJs in the extensor digitorum longus (EDL) at 8 (A,C) and 16 days post-nerve crush injury (DPI). Motor axons were labeled with YFP (green), and nicotinic acetylcholine receptors (nAChRs) were labeled with fluorescently conjugated α-bungarotoxin (fBTX, red). (E) The percentage of innervated NMJs, either fully, partially, or not at all. (F–H) Morphological analysis of NMJs, including (F) the presence of axon sprouts, (G) multiple innervations, and (H) nAChR fragmentation. * p < 0.05 versus injury-matched control; ‡ genotype effect, p < 0.05; two-way ANOVA with Šídák’s multiple comparisons test. n = 3. All values are mean ± SD. Scale bar = 25 µm.

Article Snippet: For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD.

Techniques: Labeling, Control

Journal: International Journal of Molecular Sciences

Article Title: Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing

doi: 10.3390/ijms23052572

Figure Lengend Snippet: Significantly altered functional classes of proteins during stromal cell differentiation. Panther, KEGG and Reactome pathway analysis of the significantly altered proteins revealed actin cytoskeleton regulation, integrin signaling pathway, ROBO receptor signaling proteins and extracellular matrix proteins as the most significantly altered pathways in corneal stromal cell differentiation. ( I ) Integrin signaling. (a–c) represent the expression of Rap-1b, F and TLN in fibroblasts and myofibroblasts across all biological replicates. (d) Data represented as standard deviation from all four biological replicates, Ras-related protein (HCF (FC = 0.91), MYO (FC = 0.77)), fibronectin (HCF (FC = 1.07), MYO (FC = 1.31)), talin (HCF (FC = 1.20) and MYO (FC = 1.20)). ( II ) Extracellular matrix proteoglycan proteins. (a–e) represent the expression of SERPINH1, OSN, CTSB, ITGB1 and CRTAP in fibroblasts and myofibroblasts across all biological replicates. (f) Data represented as standard deviation from all four biological replicates, serpin H1 (HCF (FC = 1.28), MYO (FC = 1.38)), osteonectin (HCF (FC = 1.41), MYO (FC = 1.78)), cathepsin B (HCF (FC = 0.66), MYO (FC = 0.70)), integrin beta-1 (HCF (FC = 1.13), MYO (FC = 1.33)) and cartilage-associated protein (HCF (FC = 1.21), MYO (FC = 1.25)). ( III ) Actin cytoskeletal-related proteins. (a–e) represent the expression of COF, LAMB1, CNN2, ANXA2 and MSN in fibroblasts and myofibroblasts across all biological replicates. (f) Data represented as standard deviation from all four biological replicates, cofilin (HCF (FC = 1.35), MYO (FC = 1.57)), lamin B1 (HCF (FC = 0.82), MYO (FC = 0.79)), calponin-2 (HCF (FC = 1.09), MYO (FC = 1.13)), annexin A2 (HCF (FC = 1.15), MYO (FC = 1.26)) and moesin (HCF (FC = 1.22), MYO (FC = 1.15)). ( IV ) ROBO receptor signaling proteins. (a–c) represent the expression of PFN1, CPN1 and PMSA1 in fibroblasts and myofibroblasts across all biological replicates. (d) Data represented as standard deviation from all four biological replicates, profilin 1 (HCF (FC = 1.41), MYO (FC = 1.50)), caprin-1 (HCF (FC = 1.63), MYO (FC = 1.70)) and proteasome subunit alpha type 1 (HCF (FC = 1.06), MYO (FC = 1.18)).

Article Snippet: For validation of the differentially altered proteins by Western blotting, the following antibodies were used: rabbit monoclonal anti-profilin 1 (1:10,000, ab124904, Abcam, Cambridge, UK), mouse monoclonal anti-cofilin (1:1000, ab54532, Abcam, Cambridge, UK) and rabbit polyclonal anti-annexin A2 (1: 1000, ab41803, Abcam, Cambridge, UK).

Techniques: Functional Assay, Cell Differentiation, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing

doi: 10.3390/ijms23052572

Figure Lengend Snippet: ( A ) (i) Western blotting: Ten micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with actin cytoskeletal proteins cofilin (COF), profilin 1 (PROF1) and annexin A2 (ANXA2) in keratocytes, fibroblasts and myofibroblasts as normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (ii–iv) Densitometry analysis of proteins: Quantification levels of cofilin, profilin 1 and annexin A2 in KCT, HCF and MYO cell lines. All proteins have been analyzed on the same gel. ( B ) (I–IV) Immunohistochemical analysis of profilin 1, cofilin, filamin A and cathepsin B. Data are from a representative experiment including the fibrotic and non-fibrotic areas of patient cornea and a control cornea. (I) Profilin 1 was predominantly expressed in stroma of viral keratitis and fungal keratitis group and to some extent in stroma of ulcer group. Higher profilin 1 expression in the fibrotic stroma is the representative myofibroblastic activation of stromal fibrocytes as shown in the top lane. Control group did not show any expression of profilin 1. (II). Filamin A expression was significantly higher in stroma of fungal keratitis group as compared to the control. Higher expression of filamin A in stromal scarred tissue indicates the presence of myofibroblast-mediated stromal fibrosis. (III) Cofilin showed higher expression in fibrotic stroma of all groups (viral keratitis, ulcer and fungal keratitis) as compared to the control. Higher expression of cofilin indicates presence of mature myofibroblasts in the scarred corneal stroma. (IV). Cathepsin B expression was significantly higher in stroma of fungal keratitis, viral keratitis and ulcer as compared to the control. Higher cathepsin B expression indicates myofibroblastic activation of stromal fibrocytes. The arrow represents the scarred area in the tissue as shown by positive staining.

Article Snippet: For validation of the differentially altered proteins by Western blotting, the following antibodies were used: rabbit monoclonal anti-profilin 1 (1:10,000, ab124904, Abcam, Cambridge, UK), mouse monoclonal anti-cofilin (1:1000, ab54532, Abcam, Cambridge, UK) and rabbit polyclonal anti-annexin A2 (1: 1000, ab41803, Abcam, Cambridge, UK).

Techniques: Western Blot, Immunohistochemical staining, Expressing, Activation Assay, Staining

Journal: Advanced Science

Article Title: Actomyosin Activity and Piezo1 Activity Synergistically Drive Urinary System Fibroblast Activation

doi: 10.1002/advs.202303369

Figure Lengend Snippet: Stiffness regulates bladder FMT via NMIIA‐mediated cellular traction force. A–C) Increased stiffness enhanced the p‐NMIIA and Lamin A/C labeling intensity ( n = 50). D–F) WB analysis shows the increased stiffness induced the expression of p‐NMIIA and Lamin A/C. G–I) Increased stiffness enhanced the Col1 and α‐SMA labeling intensity ( n = 50). J–L) WB analysis shows the increased stiffness induced Col1 and α‐SMA expression. M–O) Blebb decreased the p‐NMIIA and Lamin A/C labeling intensity. P–R) WB analysis shows that Blebb decreased the p‐NMIIA and Lamin A/C expression. S–U) Blebb decreased the Col1 and α‐SMA labeling intensity. V–X) WB analysis shows that Blebb decreased the Col1 and α‐SMA expression. Scale bar = 20 µm; * p < 0.05, ** p < 0.01; Shown is the mean ± SD.

Article Snippet: Bladder specimens and cells were fixed, permeabilized, and labeled for filamentous actin (F‐actin) (2219253, 1:500 dilution) from Invitrogen; Piezo1 (APC087, 1:200 dilution) from Alomone Laboratories, p‐FAK (ab81298, 1:200 dilution), RhoA (ab187027, 1:200 dilution), ROCK (ab97592, 1:200 dilution), and Col1 (ab138492, 1:200 dilution) from Abcam; α‐SMA (48938, 1:200 dilution), p‐PI3K (17366, 1:200 dilution) and p‐Myosin (14611S, 1:200 dilution) from Cell Signaling Technologies; Lamin A/C (NB100‐74451, 1:200 dilution) from NOVUS at 4 °C overnight.

Techniques: Labeling, Expressing

Journal: Advanced Science

Article Title: Actomyosin Activity and Piezo1 Activity Synergistically Drive Urinary System Fibroblast Activation

doi: 10.1002/advs.202303369

Figure Lengend Snippet: Piezo1 mediates stiff ECM‐induced bladder FMT. A) KEGG enrichment analysis of the DEGs between the sham‐operated mice bladder and BOO mice bladder indicated that the calcium ion pathway was chiefly responsible for BOO‐induced bladder remodeling ( n = 3). B) Cluster analysis of all ion channels in the transcriptome sequencing results. C) Cluster analysis of all ion channels in the transcriptome sequencing results show that Piezo1 messenger RNA levels were the most abundantly expressed channel and it was prominently upregulated in the BOO bladder. D) Piezo subtypes expression in sham‐operated mice bladder and BOO mice bladder were detected by WB and quantification(blow) shown in (E) ( n = 3). F) Immunofluorescence was performed to co‐localize cellular traction force markers and activated fibroblast markers ( n = 3). Scale bar = 200 µm. G–I) Inhibition of Piezo1 by siRNA was confirmed at functional (Ca 2+ influx) and biochemical (IF) levels; Scale bar = 20 µm. J–L) Inhibition of Piezo1 by siRNA decreased the Col1 and α‐SMA intensity ( n = 50); Scale bar = 20 µm. * p < 0.05, ** p < 0.01;Shown is the mean ± SD.

Article Snippet: Bladder specimens and cells were fixed, permeabilized, and labeled for filamentous actin (F‐actin) (2219253, 1:500 dilution) from Invitrogen; Piezo1 (APC087, 1:200 dilution) from Alomone Laboratories, p‐FAK (ab81298, 1:200 dilution), RhoA (ab187027, 1:200 dilution), ROCK (ab97592, 1:200 dilution), and Col1 (ab138492, 1:200 dilution) from Abcam; α‐SMA (48938, 1:200 dilution), p‐PI3K (17366, 1:200 dilution) and p‐Myosin (14611S, 1:200 dilution) from Cell Signaling Technologies; Lamin A/C (NB100‐74451, 1:200 dilution) from NOVUS at 4 °C overnight.

Techniques: Sequencing, Expressing, Immunofluorescence, Inhibition, Functional Assay

Journal: Disease Models & Mechanisms

Article Title: The glucocorticoid receptor acts locally to protect dystrophic muscle and heart during disease

doi: 10.1242/dmm.050397

Figure Lengend Snippet: Deletion of the GR worsens dystrophic cardiomyopathy. (A) qRT-PCR confirmed knockout of the GR in the mouse heart. (B) Functional knockout of the GR was confirmed by assaying a known cardiomyocyte GR target gene ( Ptgds ). (C,D) Deletion of the GR in dKO versus mdx52 mice resulted in visibly enlarged hearts (C) and a significant increase in heart mass (D). Scale bars: 4 mm. (E) The hypertrophy genes Acta2 and Myh7 were significantly upregulated in dKO hearts. (F) The inflammatory transcripts Ccl2 and Il6 were significantly upregulated specifically in dKO hearts. (G,H) Echocardiography of aged mice showed a worsening of dystrophic cardiomyopathy in 1-year-old dKO versus mdx52 mice ( n ≥4 per group). (G) Representative M-mode images of the parasternal short axis. (H) Quantification of heart function via the ejection fraction demonstrates systolic dysfunction (left). Left ventricular (LV) wall thickness (right) measured at diastole showed an increase for dKO mice. n ≥6 mice per group unless otherwise specified. Data show mean±s.e.m. (significant outlier removed from F after ROUT test). * P ≤0.05; ** P ≤0.005; *** P ≤0.0005; **** P ≤0.0001; unpaired two-tailed t -test of Cre-positive versus control littermate genotypes.

Article Snippet: The assay IDs were: Cre recombinase, Mr00635245_cn; GR ( Nr3c1 ), Mm00433832_m1; dystrophin at its 3′ end, 00464531_m1; Ccl2 , Mm00441242_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Tlr7 , Mm00446590_m1; Ptgds , Mm01330613_m1; Acta2 , Mm01546133_m1; Acta1 , Mm00808218_g1; Myh7 , Mm00600555_m1; Hprt , Mm01545399_m1; and 18S rRNA, Mm03928990_g1. qRT-PCR data were normalized to the geometric mean of the levels of the control Hprt gene and 18S rRNA. miRNAs were quantified using individual TaqMan assays on the QuantStudio 7 real-time PCR machine as previously described ( ).

Techniques: Quantitative RT-PCR, Knock-Out, Functional Assay, Two Tailed Test, Control