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: 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

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: Progranulin Promotes the Formation and Development of Capsules Caused by Silicone in Sprague-Dawley Rats

doi: 10.2147/CCID.S374128

Figure Lengend Snippet: R-PGRN promotes the functional activation and differentiation of fibroblasts by activating the TGF-β/SMAD signaling pathway. ( A ) The expression of collagen types I (COL-I) and III (COL-III), α-SMA, TGF-β1 and p-SMAD2&3 in different groups were detected by Western blot analysis. Actin served as a control. ( B ) The expressions of COL-I and COL-III, α-SMA, TGF-β1 and p-SMAD2&3 in different groups were detected by Western blot analysis after pretreatment with HY-10431 or not. GAPDH served as a control. Data are shown as the mean ± SEM (n = 3) (*p < 0.05 vs control).

Article Snippet: Primary antibodies against the following proteins were used: GAPDH (1:500, Waneibio), β-actin (1:500, Waneibio), collagen I (1:500, Waneibio), collagen III (1:500, Waneibio), MMP-1 (1:500, Waneibio), p-SMAD2&3 (1:500, Waneibio), SMAD2&3 (1:500, Waneibio), α-SMA (1:500, Waneibio): 500, Waneibio), BCL-2 (1:500, Waneibio), BAX (1:500, Waneibio), TGF-β1 (1:500, Waneibio), PGRN (1:500, Biorbyt, UK) and TIMP-1 (1:500, Waneibio).

Techniques: Functional Assay, Activation Assay, Expressing, Western Blot, Control

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: Progranulin Promotes the Formation and Development of Capsules Caused by Silicone in Sprague-Dawley Rats

doi: 10.2147/CCID.S374128

Figure Lengend Snippet: Increasing the local PGRN content promotes capsular formation and contracture. ( A ) The implant and the fibrous capsule on it at day 112 after operation. ( B ) H&E and Masson’s trichrome staining. The collagen in the capsule were stained by Masson’s trichrome (blue) (scale bar = 500 µm). ( C ) The expression levels of proteins COL-I, α-SMA, MMP-1, TIMP-1, TGF-β1 and p-SMAD2&3 in capsular tissues in different groups were detected by Western blot analysis. GAPDH served as a control. Data are shown as the mean ±SEM (n = 3) (*p < 0.05 vs control).

Article Snippet: Primary antibodies against the following proteins were used: GAPDH (1:500, Waneibio), β-actin (1:500, Waneibio), collagen I (1:500, Waneibio), collagen III (1:500, Waneibio), MMP-1 (1:500, Waneibio), p-SMAD2&3 (1:500, Waneibio), SMAD2&3 (1:500, Waneibio), α-SMA (1:500, Waneibio): 500, Waneibio), BCL-2 (1:500, Waneibio), BAX (1:500, Waneibio), TGF-β1 (1:500, Waneibio), PGRN (1:500, Biorbyt, UK) and TIMP-1 (1:500, Waneibio).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

doi: 10.1186/s13046-019-1211-2

Figure Lengend Snippet: Vascular morphology, maturity and functional status of the metastatic breast cancers. 4 T1 and MDA-MB-231 metastatic breast cancer cells were orthotopically injected into the fat pad of mice. ( a ) 3D-reconstruction of CD31 + vessels in 4 T1 and MDA-MB-231 (MM-231) tumors from untreated mice. Tumor sections were stained with an anti-CD31 antibody. Bars: 100 μm. ( b ) Immunofluorescent double-staining for PCNA (Green) and CD31 (Red) in sections of untreated CT-26 tumors. White arrows indicate PCNA nucleus positive endothelial cells that are proliferating. Nuclei were counterstained with DAPI. Bars: 100 μm. ( c ) Double immunostaining (Left) for CD31 (Green) and α-SMA (Red) in frozen sections of normal breast and 4 T1 tumors, and quantification (right) of percentage of α-SMA + /CD31 + vessels (of CD31 + vessels; n = 8). White and yellow triangles indicate disassociated vascular smooth muscle cells (VSMCs) and associated VSMCs, respectively. Nuclei were counterstained with DAPI. Bars: 100 μm. ( d ) Double immunostaining for CD31 (Green) and NG-2 (Red) in frozen sections of untreated 4 T1 tumors. White triangle indicates the pericyte associated with the vessel wall. Bars: 100 μm. ( e ) Representative images showing FITC-conjugated dextran perfused CD31 + vessels (Red) in 4 T1 tumors from untreated mice. FITC-conjugated dextran was injected through the tail vein in advance. White triangles indicate CD31 + vascular lumen containing FITC-Dextran; white arrows indicate dextran leaking outside the vessel wall. Bars: 100 μm. ( f ) Representative image for showing Pimonidazole (PIMO; brown) staining, and quantification of PIMO + hypoxic areas in 4 T1 tumors and normal breast of BALB/c mice (Lower Right Corner, n = 8). Bars: 100 μm. Quantitative data are indicated as mean ± SEM. *** p < 0.001

Article Snippet: Primary antibodies: CD31 (anti-rabbit, ab28364, Abcam; anti-Rat, ab7388, Abcam), PDGF-B (BA0519–2, Boster), α-SMA (BM0002, Boster), VE-cadherin (No.138101, BioLegend), cl-PARP (#9542, Cell Signaling), PCNA (BM3888, Boster), cl-Caspase-9 (#9542, Cell Signaling), cl-Caspase-3 (#9661, Cell Signaling); NG-2 (R&D, MAB6689).

Techniques: Functional Assay, Injection, Staining, Double Staining, Double Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

doi: 10.1186/s13046-019-1211-2

Figure Lengend Snippet: Effects of the metformin administration on angiogenesis and the vascular maturity. ( a ) CD31 immunostaining in sections of 4T1 tumors from mice untreated or treated with metformin at concentrations of 25 mg/kg•day or 225 mg/kg•day. White arrows indicate vascular sprouts of microvessels. Bars: 100 μm. (B and C) quantifications of vascular ( b ) sprouts and ( c ) branches (per mm 2 ; n = 8). ( d ) Quantification of mean fluorescent intensity of CD31 signaling of vessels in 4 T1 tumors (n = 8), revealing the unaffected CD31 expression level of vessels. ( e ) Analyzes of diameter distribution of vessels in 4T1 tumors from untreated or metformin-treated mice ( n = 8). ( f ) Double immunostaining for CD31 (Green) and α-SMA (Red) in frozen sections of 4T1 tumors from untreated or metformin-treated mice, and ( g ) quantification of percentage of α-SMA + CD31 + vessels (of CD31 + vessels; n = 8). White arrows indicate vessels with VSMCs coverage; yellows arrows indicate VSMCs disassociated with vessels. Bars: 100 μm. ( h ) Double immunostaining for CD31 and VE-cadherin of 4T1 tumor sections, revealing continuous and more abundant in metformin-treated than untreated tumors. Bars: 50 μm. ( i ) Double staining for CD31 (green) and NG-2 (red), revealing more vessels with pericyte coverage in metformin-treated than untreated MDA-MB-231 (MM-231) tumors. White and yellow arrows indicate pericytes disassociated and associated with vessels, respectively; white triangles indicate pericytes that are partially associated with vessels. Bars: 100 μm. ( j ) Quantification of NG-2 + CD31 + vessels (of CD31 + vessels) in MDA-MB-231 tumors ( n = 8). Quantitative data are indicated as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Primary antibodies: CD31 (anti-rabbit, ab28364, Abcam; anti-Rat, ab7388, Abcam), PDGF-B (BA0519–2, Boster), α-SMA (BM0002, Boster), VE-cadherin (No.138101, BioLegend), cl-PARP (#9542, Cell Signaling), PCNA (BM3888, Boster), cl-Caspase-9 (#9542, Cell Signaling), cl-Caspase-3 (#9661, Cell Signaling); NG-2 (R&D, MAB6689).

Techniques: Immunostaining, Expressing, Double Immunostaining, Double Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

doi: 10.1186/s13046-019-1211-2

Figure Lengend Snippet: Downregulation of PDGF-B by the metformin treatment. ( a ) Immunoblotting for cancer cell-derived angiogenic factors using mouse angiogenesis arrays, and ( b ) quantification of grey intensity of each factor (independent of 3 experiments). Cancer cells were treated with metformin (100 μM) for 48 h. ( c ) Column chart for showing the transcriptional levels of angiogenesis-related factors. FPKM indicates Fragments Per Kilobase of transcript per Million fragments mapped. Met indicates metformin; Met1, Met2 and Met3 indicate different reports of data of independent tumors in metformin group. ( d ) Heatmap analysis of the effect of metformin on transcriptional levels of top 9 angiogenesis-related genes of 4T1 tumors ( n = 3). The fold change ranges from − 0.5 to 0.5. “Sig.” indicates “Significance”. ( e ) Double immunostaining for CD31 and PDGF-B in 4T1 orthotopically implanted tumors (Left). shRNA-PDGF-B- and shRNA-Control-transfected 4T1 tumor-bearing mice received control or metformin treatment (225 mg/kg•day). Quantification of PDGF-B fluorescent intensity and microvessel density (Right; n = 8). ( f ) Immunostaining for CD31 and α-SMA in 4T1 cancers, revealing increased percentage of patent microarteries (n = 8). White arrows indicate the compressed lumen of arteries distributed in 4T1 cancer stroma. ( g - h ) Survival curve for analyzing the association of PDGF-B mRNA expression in primary breast tumor with survival probability of patients with breast cancer ( g ). PDGF-B mRNA levels was positively correlated with mRNA level of CD31, but not PCNA and Ki-67. Data was obtained from TCGA dataset. 1064 samples were included. FPKM: Fragments Per Kilobase of transcript per Million fragments mapped. Quantitative data are indicated as mean ± SEM. “ns” indicates not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Primary antibodies: CD31 (anti-rabbit, ab28364, Abcam; anti-Rat, ab7388, Abcam), PDGF-B (BA0519–2, Boster), α-SMA (BM0002, Boster), VE-cadherin (No.138101, BioLegend), cl-PARP (#9542, Cell Signaling), PCNA (BM3888, Boster), cl-Caspase-9 (#9542, Cell Signaling), cl-Caspase-3 (#9661, Cell Signaling); NG-2 (R&D, MAB6689).

Techniques: Western Blot, Derivative Assay, Double Immunostaining, shRNA, Control, Transfection, Immunostaining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

doi: 10.1186/s13046-019-1211-2

Figure Lengend Snippet: Effects of PDGF-B knockdown on metastasis, chemosensitization and vascular maturity and functionality. ( a ) Double staining for CD31 (green) and α-SMA (red) of 4T1 tumor sections. shRNA-PDGF-B- and shRNA-Control-transfected 4 T1 tumor-bearing mice received control, metformin (225 mg/kg), imatinib (60 mg/kg) for the combined treatment. Fluorescent signaling of series thin-layer scanning were reconstructed for 3D observation of VSMCs on vessels. VSMCs No. is at the bottom of the panel and indicated as mean ± SEM (n = 8). ( b ) Quantification of ratio of α-SMA + area/CD31 + area (upper), CD31 + area per μm 2 (middle) and percentage of α-SMA + VSMCs associated with vessels (bottom) in 4 T1 cancers (n = 8). ( c ) Representative images showing decreased vascular leakage in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors. Fitc-dextran (green) was injected through tail vein 10 mins before sacrifice. Tumor sections was counterstained with anti-CD31 antibody (Red). White arrows indicate the dextran leaking outside the vessel wall. Bars: 100 μm. ( d ) Representative images showing more lectin-perfused CD31 + vessels in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors (n = 8). Red: perfused lectin; Green: CD31 + vessels. White arrows indicate CD31 + vessels with lectin perfusion. Percentage of Lectin + /CD31 + vessels (of CD31 + vessels) is indicated as mean ± SEM (at the bottom of the panel). ( e ) H&E staining of sections of 4T1 cancers (Left) and quantification of necrotic and hemorrhagic areas (Right; n = 8). shRNA-PDGF-B or shRNA-Con-transfected 4T1 tumor-bearing mice received low dose CTX (20 mg/kg•day). “N” indicates “necrosis”; black arrows indicate tumor hemorrhage. ( f ) Decreased primary tumor lung metastasis in mice bearing shRNA-PDGF-B 4T1 cancer cells than that in mice bearing shRNA-Con 4T1 cells. (Upper) H&E staining for 4T1 tumor sections; (Lower) quantification of 4T1 metastatic index (metastatic nodules per gram of primary tumor; n = 8). Red asterisk indicates lung metastasis nodule. Magnification: 200Х. ( g ) Schematic diagram of metformin-induced vascular remodeling in metastatic breast cancers. Metastatic breast cancers are angiogenic with hypoperfusion and vascular immaturity, which contribute to vascular leakage, chemoresistance, hypoxia and distant metastasis. By decreasing PDGF-B of those metastatic breast cancers, metformin inhibits angiogenesis and improved the vascular maturity and functionality, therefore improving the chemosensitivity and reducing the distant metastasis

Article Snippet: Primary antibodies: CD31 (anti-rabbit, ab28364, Abcam; anti-Rat, ab7388, Abcam), PDGF-B (BA0519–2, Boster), α-SMA (BM0002, Boster), VE-cadherin (No.138101, BioLegend), cl-PARP (#9542, Cell Signaling), PCNA (BM3888, Boster), cl-Caspase-9 (#9542, Cell Signaling), cl-Caspase-3 (#9661, Cell Signaling); NG-2 (R&D, MAB6689).

Techniques: Knockdown, Double Staining, shRNA, Control, Transfection, Injection, Staining

Journal: Cells

Article Title: Role of Kindlin-2-Expressing Extracellular Vesicles in the Invasiveness of Triple Negative Breast Cancer Tumor Cells

doi: 10.3390/cells14131034

Figure Lengend Snippet: Autologous and heterologous effects of EV-associated K2 on tumor invasion and fibroblast activation. ( A , B ) Three-dimensional tumor spheroid invasion assays using 231 cells in the presence of either conditioned media (CM) ( A ) or purified small extracellular vesicles (EVs) ( B ) from parental or K2-knockout (K2-KO) 231 cells. Red asterisks indicate sites of invasion into the surrounding Matrigel. Quantification (right panels) shows a significant reduction in invasive potential with K2-KO CM or EVs. **, p < 0.01. Scale bars, 100 µm. ( C , D ) Western blot analysis of α-smooth muscle actin (αSMA) and fibroblast activation protein (FAP) expression in mouse 3T3 or human WI-38 fibroblasts following treatment with CM ( C ) or EVs ( D ) from parental or K2-KO 4T1 or 231 cells. Densitometric values normalized to β-actin are shown below each αSMA and FAP band, indicating robust fibroblast activation by parental CM/EVs and markedly reduced activation by K2-KO-derived counterparts. For uncropped blots, see below. ( E , F ) Functional consequences of fibroblast education by EVs on tumor invasiveness. CM from naïve or EV-educated fibroblasts were applied to 231 ( E ) or 4T1 ( F ) tumor spheroids in 3D invasion assays. Fibroblast education was performed by pre-treatment with EVs from 231 ( E ) or 4T1 ( F ) parental cells. Red asterisks mark invasive protrusions. Quantification (right panels) demonstrates enhanced invasion in the presence of CM from EV-educated fibroblasts. **, p < 0.01. Scale bars, 100 µm.

Article Snippet: The membranes were blocked for 1 h at RT in PBS containing 1% BSA, then incubated overnight at 4 °C with primary antibodies targeting human FAP (#66562, Cell Signaling, Danvers, MA, USA 1:1000), mouse FAP (#EWI042, Kerafast, Boston, MA, USA, 1:1000), Kindlin-2 (#MAB2617, EMD Millipore, 1:2000), β-Actin (#A2228, Sigma-Aldrich, 1:5000), calnexin (#MA3-027, clone AF18, Invitrogen, 1:500), CD9 (#SC-20048, clone P1/33/2, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), CD81 (#MA5-13548, clone 1.3.3.22, Invitrogen, 1:500), or α-SMA (#19245S, Cell Signaling, 1:1000).

Techniques: Activation Assay, Purification, Knock-Out, Western Blot, Expressing, Derivative Assay, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

doi: 10.1016/j.jbc.2024.107516

Figure Lengend Snippet: Renal functional impairment and pathology in mice with adriamycin nephropathy. A , albuminuria expressed as ACR (mg/mg) was evaluated in spot urine samples at baseline and weekly intervals thereafter until week 6 of ADR nephropathy. B – E , plasma levels of albumin ( B ), creatinine ( C ), urea ( D ), and cholesterol ( E ) were determined at week 2, 4, and 6 in ADR-treated mice. F , ADR-treated mice developed severe glomerular lesions with segmental to global sclerosis, tubular injury, and interstitial fibrosis indicated by Periodic acid–Schiff-positive glomerular deposits. G , Western blot analysis of KIM-1, NGAL, and α-SMA in tubulointerstitial tissues of mice treated with ADR. H , RT-PCR analysis of KIM-1, NGAL, and α-SMA in tubulointerstitial tissues of mice treated with ADR. I , immunohistochemical staining of KIM-1, COL-1, α-SMA, and E-cadherin in renal tissues of mice treated with ADR. For statistical analysis, one-way ANOVA was used for A , and a 2-tailed Student’s t test was used for B – E , G , and H . ∗ p < 0.05 compared with control mice. Scale bars, 50 μm ( F and I ). Col-1, collagen I.

Article Snippet: The membranes were incubated overnight at 4 °C using antibodies against TSP-1 (1:200), ITGB3 (1:1000), KIM-1 (1:500), Col-1 (1:1000), caspase 3 (1:2000), α-SMA (1:2000), E-cadherin (1:1000), GAPDH (1:10,000), and β-actin (1:5000) and then incubated with secondary antibodies (1:5000, Proteintech) for 2 h at 37 °C.

Techniques: Functional Assay, Clinical Proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

doi: 10.1016/j.jbc.2024.107516

Figure Lengend Snippet: THBS1 deficiency reduces albuminuria and renal functional impairment in mice with ADR nephropathy. A , level of Albuminuria (ug/mg) in WT and THBS1-KO mice treated with ADR. B – E , plasma levels of albumin, creatinine, urea, and cholesterol were determined at week 6 in WT and THBS1-KO mice treated with ADR. F , periodic acid–Schiff staining of renal sections in WT and THBS1-KO mice treated with ADR. G , Western blot analysis of TSP-1, KIM-1, and α-SMA in tubulointerstitial tissues of WT and THBS1-KO mice treated with ADR. For statistical analysis, one-way ANOVA was used for A and B . ∗ p <0.05 compared with control WT mice; # p < 0.05 compared with ADR-treated mice. Original magnifications: ×200, the scale bars represent 50 μm ( F ). ADR, adriamycin; TSP-1, thrombospondin 1.

Article Snippet: The membranes were incubated overnight at 4 °C using antibodies against TSP-1 (1:200), ITGB3 (1:1000), KIM-1 (1:500), Col-1 (1:1000), caspase 3 (1:2000), α-SMA (1:2000), E-cadherin (1:1000), GAPDH (1:10,000), and β-actin (1:5000) and then incubated with secondary antibodies (1:5000, Proteintech) for 2 h at 37 °C.

Techniques: Functional Assay, Clinical Proteomics, Staining, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

doi: 10.1016/j.jbc.2024.107516

Figure Lengend Snippet: Effect of ITGB3 inhibition by cyclo-RGDfK on the tubulointerstitial injury of ADR-treated mice. A , level of albuminuria (ug/mg) in ADR- and cyclo-RGDfK–treated mice. B – E , serum levels of albumin, creatinine, urea, and cholesterol were determined at week 6 in mice treated with ADR and cyclo-RGDfK. F , PAS and Masson staining of renal sections in mice treated with ADR and cyclo-RGDfK. G , immunohistochemical staining of TSP-1, ITGB3, KIM-1, E-cadherin, COL-1, α-SMA, and F4/80 in tubulointerstitial tissues of mice treated with ADR and cyclo-RGDfK. H , Western blot analysis of TSP-1, ITGB3, KIM-1, NGAL, E-cadherin, α-SMA, and COL-1 in tubulointerstitial tissues of mice treated with ADR and cyclo-RGDfK. I , RT-PCR analysis of THBS1, ITGB3, NGAL, KIM-1, α-SMA, IL-6, TGF-β, IL-1β, COL-1, and fibronectin in tubulointerstitial tissues of mice treated with ADR and cyclo-RGDfK. For statistical analysis, one-way ANOVA was used for A , B , H , and I . ∗ p < 0.05 compared with control mice. # p <0.05 compared with ADR-treated mice. Original magnifications: ×200, the scale bars represent 50 μm ( F and G ). ADR, adriamycin; BSA, bovine serum albumin; Col-1, collagen I; ITGB3, integrin β3; TGF-β, transforming growth factor-β; TSP-1, thrombospondin 1.

Article Snippet: The membranes were incubated overnight at 4 °C using antibodies against TSP-1 (1:200), ITGB3 (1:1000), KIM-1 (1:500), Col-1 (1:1000), caspase 3 (1:2000), α-SMA (1:2000), E-cadherin (1:1000), GAPDH (1:10,000), and β-actin (1:5000) and then incubated with secondary antibodies (1:5000, Proteintech) for 2 h at 37 °C.

Techniques: Inhibition, Staining, Immunohistochemical staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: The Journal of Biological Chemistry

Article Title: Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

doi: 10.1016/j.jbc.2024.107516

Figure Lengend Snippet: Effect of ITGB3 inhibition by cyclo-RGDfK on HK-2 cells injury. A , verification of ITGB3 knockdown in HK-2 cells. RT-PCR analysis of ITGB3 ( A ) and Western blot analysis of integrinβ3 ( B ) in HK-2 cells treated with ITGB3-siRNA. C , Western blot analysis of integrinβ3, TSP-1, and KIM-1 in HK-2 cells treated with ITGB3-siRNA exposed to rTSP-1 (1 ug/ml) for 24 h. D , Western blot analysis of integrinβ3, TSP-1, and KIM-1 in HK-2 cells treated with ITGB3-siRNA exposed to BSA (20 mg/ml) for 24 h. E , Western blot analysis of integrinβ3, TSP-1, KIM-1, and α-SMA in HK-2 cells treated with cyclo-RGDfK (0.5 uM) and rTSP-1 (1 ug/ml) for 24 h. For statistical analysis, one-way ANOVA was used for A – C . ∗ p < 0.05 compared with control cells. # p < 0.05 compared with cells exposed to BSA (20 mg/ml) or rTSP-1 (1 ug/ml). BSA, bovine serum albumin; Col-1, collagen I; rTSP-1, recombinant human TSP-1 protein; TSP-1, thrombospondin 1; ITGB3, integrin β3.

Article Snippet: The membranes were incubated overnight at 4 °C using antibodies against TSP-1 (1:200), ITGB3 (1:1000), KIM-1 (1:500), Col-1 (1:1000), caspase 3 (1:2000), α-SMA (1:2000), E-cadherin (1:1000), GAPDH (1:10,000), and β-actin (1:5000) and then incubated with secondary antibodies (1:5000, Proteintech) for 2 h at 37 °C.

Techniques: Inhibition, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Recombinant