Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 2. Knockout of HSP90b in MODE-K cells eliminates the accumulation of stachyose on the cell membrane and the regulatory effects of stachyose on the exosomal miRNA expression (A–C) Identification of exosome-sized extracellular vesicles secreted by MODE-K cells. The data were representative of three independent experiments. See also Figure S2B. (A) Morphology of exosomes secreted by MODE-K cells treated with 2 mM of stachyose was visualized by electron microscopy on the 100-mesh copper mesh (with carbon film). Representative from three experiments. Scale bar, 100 nm. (B) Nanoparticle tracking analysis measured by NanoSight. (C) The protein levels of Tsg 101 and CD 63 in exosomes were measured by western blotting. (D–F) Profiling of exosomal miRNAs released by MODE-K cells treated with 0 (Sta0), 2 mM (Sta2), or 4 mM (Sta4) stachyose based on small RNA sequencing results (n = 4). (D) PCA was performed using the OmicStudio tools. p value and R value were calculated by vegan package of R. PCA was performed by stats package. Figures were drawn by ggplot2 package. (E) Volcano plots of miRNA levels in stachyose-treated versus non-treated MODE-K cells. (F) Heatmap of highly expressed miRNAs selected from all detected 327 mouse miRNAs. Highly expressed miRNAs were identified as relative expression > 203.3 (mean relative expression of all miRNAs). (G) The subcellular localization of stachyose on the HSP90b-WT and HSP90b-KO MODE-K cells. Scale bar, 10 mm. (H) The relative expression levels of 12 miRNAs in the exosomes derived from HSP90b-WT and HSP90b-KO MODE-K cells after treated with 0, 2, and 4 mM stachyose. *p < 0.05.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Knock-Out, Membrane, Expressing, Electron Microscopy, Western Blot, RNA Sequencing, Derivative Assay

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 3. Stachyose reconstitutes fecal miRNA profiles in mice and humans (A and B) Fecal miRNA profile of mice receiving 0 (NC), 200 (S-L), 400 (S-M), and 800 (S-H) mg/kg$bw stachyose tested by small RNA sequencing analysis (n = 4). (A) Heatmap of highly expressed miRNAs selected from all detected 107 mouse miRNAs. Highly expressed miRNAs were identified as relative expres- sion > 156.34 (mean relative expression of all miRNAs). (B) Differentially expressed miRNAs filtered by Log2FC of relative expression and expression variation (| Log2FC|>1, miRNA expression variation > 50, the expression variation was defined as the absolute differences in the relative expression levels between two groups). The significant ones (p < 0.05) were emphasized in yellow. (C) Relative expression of miR-191-5p, miR-30a-5p, and miR-21a-5p. Data were represented as mean ± SEM (n = 3). *p < 0.05. (D–F) Fecal miRNA profile of healthy human subjects (six males and six females). Feces from 12 human subjects were collected pre and post stachyose (5 g/kg$bw, recommended daily intake) administration. (D) Clinical trial protocol. All subjects received stachyose supplementation for 8 weeks. (E) PCAs of miRNAs in human feces based on small RNA sequencing results. The method for PCA analysis was same as Figure 2. (F) Log2FC values of 28 highly expressed miRNAs (miRNA expression > 50) among all detected 138 human miRNAs. (G) UpSetR and Venn analyses of all downregulated highly expressed miRNAs detected both in mice and human feces. UpSetR analysis was performed by UpSetR package of R. Venn analysis was performed by yyplot package.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: RNA Sequencing, Expressing

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 5. The axis of stachyose-fecal miRNA-gut microbiota: Stachyose directly regulates fecal miRNAs, which, in turn, shape the gut microbiota (A) Relative expression of 16S rRNA gene in fecal samples from normal and antibiotic-treated pseudo-germ-free (PGF) mice. The relative 16S rRNA level was calculated based on Ct values tested by qPCR analysis (n = 3). *p < 0.05, **p < 0.01. (B–D) Fecal miRNA profile of stachyose-treated (Ab-S) and -untreated (Ab) PGF mice (n = 4). (B) PCA analysis of fecal miRNA levels based on small RNA sequencing results. (C) Differentially expressed miRNAs filtered by Log2FC of relative expression and expression variation (|Log2FC|>1, miRNA expression variation > 10) in PGF mice. The significant ones (p < 0.05) were emphasized in yellow. (D) Venn analysis of all downregulated highly expressed miRNAs detected both in PGF mice (Ab-S vs. Ab) and normal mice (S-L S-M S-H vs. NC) feces. The highly expressed miRNAs are shown in Figure S4H. (E–G) Gut microbiome structure of antibiotics-pretreated mice receiving fecal miRNAs harvested from mice in Ab-S or Ab group (n = 5). (E) After 4 weeks of pre- treatment with antibiotics, feces collected from donors (stachyose-treated [Ab-S] and -untreated [Ab] PGF mice) were heat inactivated, and the isolated fecal miRNAs were transplanted to recipients (miR-Ab/miR-Ab-S) for 8 weeks. (F) PCA with unweighted UniFrac distance of all detected genera in feces from miR-Ab and miR-Ab-S mice. (G) Log2FC values of significantly altered five genera with mean relative abundance > 0.5. The color represents the phylum that the genus belonged to.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Isolation

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 6. miR-30a-5p restrains the proliferation of Lactobacillus reuteri (A) Linear regression analysis on the relative expression levels of stachyose-downregulated fecal miRNAs in both normal and PGF mice (related to Figure 5D) and relative abundance of Lactobacillus genus. The data of Lactobacillus genus were based on 16S rRNA sequencing data for normal and PGF mice. Linear regression analysis was performed by GraphPad Prism with 95% confidence interval. (B) Venn analysis of all downregulated highly expressed miRNAs detected in normal mice feces, PGF mice feces, human feces, and MODE-K-cell-secreted exosomes. (C) FITC-labeled (green) miR-30a-5p and miR-191-5p were co-cultured with Lactobacillus gasseri (Lg) and Lactobacillus reuteri (Lr). Fluorescence signal was captured by fluorescence microscope (ZEISS, DMi8 automated, left). Representative from three experiments. The relative expression of miR-30a-5p and miR- 191-5p in Lg (blue fill) and Lr (yellow fill) was detected by qPCR analysis (n = 3, right). See also Figures S6D and S6E. (D) Target site sequence alignment of miR-191-5p and miR-30a-5p against Lr mRNA sequence (matching sites highlighted). The interaction energy was measured by Targetscan score and Miranda energy. (E) Lg and Lr were grown in culture media with 2-mM miRNA mimics and their scrambled controls for 24 h. Bacterial growth was monitored as absorbance at 600 nm (OD600). Representative growth curves of three independent experiments with duplicates were presented. See Figure S6F for additional growth curves. *p < 0.05 compared with vehicle, #p < 0.05 compared with scramble control. Data were represented as mean ± SEM.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Expressing, Sequencing, Labeling, Cell Culture, Fluorescence, Microscopy, Control

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 7. The crosstalk between gut microbiota and fecal miRNA (A) After 4 weeks of pre-treatment with antibiotics, fecal materials containing both miRNAs and bacteria collected from donors (mice receiving 0 [NC] and 400 [S-M] mg/kg$bw stachyose, same as Figure 4) were transplanted to recipients (Naive-NC/Naive-S) for 8 weeks. (B) Relative expression of 16S rRNA gene in feces samples from normal and antibiotics-pretreated recipient mice. The relative 16S rRNA expression was calculated by Ct value tested by qPCR analysis (n = 3). (C–E) PCA with unweighted UniFrac distance (C), genera distribution (D), and the change ratio of Lactobacillus (E) for antibiotics-pretreated miR-Ab, miR-Ab-S, Naive-NC, and Naive-S mice (n = 5).*p < 0.05, **p < 0.01. (F) Lg and Lr were grown in culture media with bacterial metabolites from miR-Ab and miR-Ab-S. Growth was monitored as absorbance at 600 nm (OD600) for 24 h. Representative growth curves of three independent experiments with duplicates were presented. *p < 0.05, **p < 0.01. Data were represented as mean ± SEM. (G and H) PCA (G) and the change ratio of miR-30a-5p (H) for antibiotics-pretreated miR-Ab, miR-Ab-S, Naive-NC, and Naive-S mice (n = 4).

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Bacteria, Expressing

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: The Journal of nutrition

Article Title: Suppression of High-Fat Diet-Induced Obesity by Platycodon Grandiflorus in Mice Is Linked to Changes in the Gut Microbiota.

doi: 10.1093/jn/nxaa159

Figure Lengend Snippet: FIGURE 2 Fatty acid catabolism and lipid anabolism in the adipose tissues of male C57BL/6J mice fed CON, HFD, or HFPG. (A) Map of genes involved in fat accumulation and fatty-acid metabolism affected by PG supplementation. (B) Western blot analysis for p-AMPK, p-ACC, and FASN in epididymal adipose tissues. (C) Fold change in densitometric concentrations of p-AMPK/AMPK, p-ACC/ACC, and FASN/ACTB relative to the CON group (n = 3). (D) Relative levels of mRNA expression in epididymal adipose tissues tested by qRT-PCR (n = 3). Values represent means ± SEMs, n = 7 if not specified. Labeled means without a common letter differ, P < 0.05. ACC, acetyl-CoA carboxylase; ACTB, actin, β; Adipoq, adiponectin; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; CEBPA, CCAAT/enhancer binding protein α; CON, control diet; FASN, fatty acid synthase; HFD, high-fat diet; HFPG, high-fat diet and PG supplementation; Lep, leptin; p-ACC, phosphorylated acetyl-CoA carboxylase; p-AMPK, phosphorylated AMP-activated protein kinase; PG, Platycodon grandiflorus; PPARG, peroxisome proliferator– activated receptor γ ; Srebf1, sterol regulatory element binding transcription factor 1; SREBP-1c, sterol regulatory element binding protein-1c; WAT, white adipose tissue.

Article Snippet: The primary antibodies against AMP-activated protein kinase α (AMPKα) (no. 5831), phosphorylated-AMPKα (p-AMPKα) (Thr172) (no. 2535), acetyl-CoA carboxylase (ACC) (no. 3676), phosphorylated-ACC (p-ACC) (Ser79) (no. 11818), fatty acid synthase (FASN) (no. 3180), and horseradish peroxidase–conjugated anti-rabbit secondary antibody (no. 7074) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Labeling, Binding Assay, Control

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: Fig. 3 MSI2 directly binds to EGFR and ERBB3 mRNA. A Quantification of mRNA immunoprecipitation (RIP) results from assays performed in A549 and PC9 cell lysates using antibodies to MSI2, or IgG (negative control) antibodies, followed by quantitative RT-PCR. Data are normalized to positive control PTP4A1, TGFBR1, and SMAD3 are additional positive controls; GAPDH is a negative control. Data shown reflect the average of three independent RIP experiments. Error bars indicate SEM. Statistical analysis was performed using unpaired two tailed t-test. p < 0.05, **p < 0.01, ***p < 0.001 for all graphs. B Location of consensus binding sites for Musashi proteins in EGFR, as defined from studies by Bennett et al.18 and Wang et al.19. Coding sequences are represented by thick lines; 3′ untranslated regions by thin line. 7- or 8-bp consensus sequences are indicated by arrows. Thick arrows indicate identical concensus sequences identified simultaneously by Wang and Bennett studies. Shorter consensus sequences are not indicated. Blue arrows indicate the positions of ssRNA oligos (MSI2-binding sites are underscored) used for REMSA. The localization of the fragments used to generate reporter vectors are depicted as Reporter 1 and Reporter 2. C Analysis of recombinant MSI2 protein binding with 3′UTR fragments of EGFR mRNA by RNA-EMSA. In all, 50 ng of recombinant MSI2 protein were incubated with 32P-labeled ssRNA oligos, EGFR oligo 1, EGFR oligo 2, and Positive- and Negative control oligos alone, or in presence of 100-fold molar excess of unlabeled competitors, identical to the labeled probe. Competing ssRNA EGFR oligos 1 and 2 were identical to labeled probes and contained wild type (oligo wt) or mutant (oligo mut) MSI2-binding motifs.

Article Snippet: The sections were incubated overnight with primary antibodies to MSI2 (EP1305Y, Rabbit, 1:100, Abcam #ab76148), EGFR (D38B1, Rabbit, 1:50, Cell signaling, Cat #4267) at 4 °C in a humidified slide chamber.

Techniques: Immunoprecipitation, Negative Control, Quantitative RT-PCR, Positive Control, Two Tailed Test, Binding Assay, Recombinant, Protein Binding, Incubation, Labeling, Mutagenesis

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 2. LC-derived Exos induce macrophage polarization toward to M2 phenotype. Notes: After PMA induction, the morphology of THP-1 cells was observed under a microscope (A) (n = 3). qRT-PCR was applied to detect the expression of macrophage surface marker CD68 (B) and surface markers of M1 (iNOS and IL-1β) and M2 (CD206, CD163 and arginase-1) (D) (n = 3). The uptake of Exos in macrophages was inspected by fluorescence labeling of Exos (C) (n = 3). The protein levels of M2 markers CD206, CD163 and arginase-1 in macrophages were assessed by Western blot (E) (n = 3); **P < 0.01, ***P < 0.001, compared to the M group; LC, lung cancer; Exos, exosomes; PMA, phorbol-12-myristate-13-acetate; M, macrophage.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Derivative Assay, Microscopy, Quantitative RT-PCR, Expressing, Marker, Fluorescence, Labeling, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 5. Exosomal circPVT1 stimulates macrophage polarization to M2 type and enhances the biological function of LC cells. Notes: The A549 cells were transfected with oecircPVT1 or si-circPVT1. Exos were extracted from the transfected A549 cells (Exo- A-oecircPVT1 or Exo-A-si-circPVT1) and coincubated with macrophages (M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1). Then, qRT- PCR was utilized to measure the mRNA level of circPVT1 in A549 cells (A) and Exo-A (B) as well as the expressions of CD206, CD163 and arginase-1 in M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 (C) (n = 3). The protein expressions of M2 markers in M+ Exo- A-oecircPVT1 or M+ Exo-A-si-circPVT1 were detected by Western blot (D) (n = 3). Subsequently, the proliferation, invasion and migration abilities of A549 cells that coincubated with M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 were determined by CCK-8 assay (E), Transwell (F) and cell scratch assay (G), respectively (n = 3). The mRNA expressions of M2 markers and miR-124-3p in the transfected macrophages were inspected by qRT-PCR (H) (n = 3); *P < 0.05, ***P < 0.001, compared to the Blank group; *P < 0.05, **P < 0.01, ***P < 0.001, compared to the M+ Exo-A-si-NC or M+ Exo-A-vector group; *P < 0.05, **P < 0.01, compared to the M/ Blank group; M, macrophage; LC, lung cancer; Exos, exosomes.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Migration, CCK-8 Assay, Wound Healing Assay, Plasmid Preparation

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 1 Effects of adipose-specific miR-146a-5p knockout on endurance exercise capacity, metabolism, and glucose homeostasis in mice. (a) The sche matic diagram for the development strategy of miR-146a-5p-knockout mice. (b) aKO mouse WAT tissue gDNA PCR result with a sequence of only 200 bp. (c) The expression of miR-146a-5p gene in BAT, iWAT, and eWAT of Flox and aKO mice (n = 6). (d) Body weight (n = 8). (e) Body composition (n = 8). (f) Representative images of body imaging. (g) Representative images of mice. (h) Representative H&E staining of iWAT, eWAT, and BAT from mice (scale bar = 50 μm). (i) Tissue weight in BAT, iWAT, eWAT, GAS, SOL, TA, and EDL of mice (n = 7). (j) Running distance at low speed (n = 6). (k) Score of weight lifting (n = 7). (l) Muscle grip strength (n = 7). (m) Representative cross sections TA fiber immunofluorescent MyHC staining (scale bar = 100 μm). (n) Frequency histogram of fiber cross-sectional area (n = 6). (o) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in the TA muscles of Flox and aKO mice (n = 6). (p, q) The protein levels and statistical analyses of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR measured by Western blot in the TA muscles of Flox and aKO mice (n = 3). (r, s) ELISA analysis for IL-6 and TNF-α in Flox and aKO mice (n = 6). (t, u) The O2 consumption (VO2) (n = 6). (v, w) RER (n = 6). (x, y) IPITT and IPGTT blood glucose changes in Flox and aKO mice (n = 8). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Knock-Out, Sequencing, Expressing, Imaging, Staining, Quantitative RT-PCR, Muscles, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 2 3T3-L1 cell-derived miR-146a-5p participates in C2C12 cell proliferation and differentiation. (a) 3T3-L1 cells were co-cultured with C2C12 cells, and the cells were grown in a transwell. (b) The expression of miR-146a-5p gene in 3T3-L1 cells following transfection with mimics and inhibitors (n = 6). (c) The expression of miR-146a-5p gene in co-cultured C2C12 cells following transfecting 3T3-L1 cells with mimics and inhibitors (n = 6). (d) CCK-8 result of co- cultured C2C12 cells (n = 9). (e, f) EdU image and statistical analyses of C2C12 cells (scale bar = 50 μm) (n = 7). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA of C2C12 cells (n = 6). (h, i) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results of C2C12 cells (n = 3). (j) RT-qPCR analysis fo MyoD, MyoG, Fbx32 and MuFR of C2C12 cells (n = 6). (k, l) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot and the statistical analyses results of C2C12 cells (n = 3). (m, n) Representative muscle fiber immunofluorescent MyHC staining of C2C12 cells (scale bar = 50 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Cell Culture, Expressing, Transfection, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 3 miR-146a-5p inhibits proliferation and promotes differentiation in C2C12 cells. (a) RT-qPCR to detect the expression of miR-146a-5p in proliferating C2C12 cells (n = 12). (b) The expression of miR-146a-5p gene in proliferating C2C12 cells after transfection with mimics and inhibitors (n = 12). (c, d) EdU image and statistical analyses of C2C12 (scale bar = 50 μm) (n = 12). (e) CCK-8 result of C2C12 (n = 8). (f, g) Cell cycle analysis of C2C12 by flow cytometry and statistical results (n = 3). (h) RT-qPCR analysis for Cyclin A2, Cyclin B1, Cyclin D1, PCNA and P21 in C2C12 (n = 6). (i, j) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results in C2C12 (n = 3). (k) The expression of miR-146a-5p gene in differentiat ing C2C12 (n = 12). (l-n) Representative muscle fiber immunofluorescent MyHC staining in C2C12 (scale bar = 100 μm) (n = 4). (o) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in C2C12 (n = 9). (p-q) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot and the statistical analyses results in C2C12 (n = 3). (r) RT-qPCR analysis for MyHC I, MyHC IIa, MyHC IIb and MyHC IIx in C2C12 (n = 9). (s) RT-qPCR analysis for IL-1β, IL-6 and TNF-α in C2C12 (n = 9). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Cell Cycle Assay, Flow Cytometry, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 4 miR-146a-5p reversed the myotube atrophy of C2C12 cells induced by aKO-WAT-Exos. (a) Electron microscopy results and nanoparticle tracking analysis was used to determine the size distribution of adipose-derived exosomes (scale bar = 200 nm). (b) Calnexin in adipose cells and Alix, TSG101, CD9, and CD63 in adipose-derived exosomes of aKO and Flox mice were detected by Western Blot. (c) The expression of miR-146a-5p gene in Flox-iWAT- Exos, aKO-iWAT-Exos, Flox-eWAT-Exos, aKO-eWAT-Exos (n = 6). (d) CCK-8 result of C2C12 cells treated with Control (PBS), Flox-iWAT-Exos, aKO-iWAT-Exos, Flox-eWAT-Exos, aKO-eWAT-Exos (n = 8). (e, f) EdU and statistical analyses image of C2C12 (scale bar = 50 μm) (n = 6). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 cells (n = 6). (h) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot in C2C12 cells (n = 3). (i-k) Representative muscle fiber immunofluorescent MyHC staining, myotube diameter, and myotube fusion rate in C2C12 cells (scale bar = 100 μm) (n = 6). (l) RT-qPCR analysis for MyoD, MyoG, Fbx32, and MuFR in differentiated C2C12 (n = 6). (m) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot in differentiated C2C12 (n = 3). (n) The expression of miR-146a-5p gene in proliferating C2C12 treated with aKO-WAT-Exos + NC and aKO-WAT-Exos + Mimics (n = 6). (o) CCK-8 result of C2C12 (n = 8). (p, q) EdU image and statistical results of C2C12 (scale bar = 50 μm) (n = 6). (r) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 cells (n = 6). (s) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 (n = 3). (t, u) Representative muscle fiber immunofluorescent MyHC staining in C2C12 (n = 4) (scale bar = 50 μm). (v) RT-qPCR analysis for MyoD, MyoG, Fbx32 and MuFR in C2C12 (n = 6). (w) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR by Western blot in C2C12 (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Electron Microscopy, Derivative Assay, Western Blot, Expressing, CCK-8 Assay, Control, Quantitative RT-PCR, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 5 WAT-derived exosomes miR-146a-5p may be involved in muscle atrophy. (a) The fluorescence signal distribution of PKH67-labeled adipose- derived exosomes in aKO organs for 24 h after TA injection. The isolated organs from left to right are as follows: heart, liver, spleen, lung, kidney, BAT, iWAT, eWAT, GAS, SOL, TA, EDL, and intestinal fat. (b) The expression of miR-146a-5p gene in aKO TA muscles 12 h after injected with aKO-Exos and Flox-Exos (n = 6). (c) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in aKO TA muscles (12 h) (n = 6). (d) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR were measured by Western blot in aKO TA muscles (12 h) (n = 3). (e) The expression of miR-146a-5p gene in aKO TA muscles 24 h after injected with aKO-Exos and Flox-Exos (n = 6). (f) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in aKO mice TA muscles (24 h) (n = 6). (g) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR were measured by Western blot in aKO TA muscles (24 h) (n = 3). (h) Body weight gain (n = 4). (i) Body composition (n = 4). (j) Representative images of body imaging. (k) Tissue weight in GAS, SOL, TA, and EDL of mice (n = 4). (l) Running distance at low speed (n = 3). (m) Score of weight lifting (n = 4). (n) Muscle grip strength (n = 4). (o) The expression of miR-146a-5p gene in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 6). (p) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 3). (q) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR measured by Western blot in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 3). (r, s) Representative cross sections TA fiber immunofluorescent MyHC staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). (t, u) Representative cross sections TA fiber immunofluorescent MyoD staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). (v, w) Representative cross sections TA fiber immunofluorescent Pax7 staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Fluorescence, Labeling, Injection, Isolation, Expressing, Muscles, Quantitative RT-PCR, Western Blot, Imaging, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 6 miR-146a-5p alleviates muscle atrophy in vitro. (a, b) Representative muscle fiber immunofluorescent MyHC staining of CTX-induced C2C12 cells after transfection with miR-146a-5p mimics/inhibitors or co-treatment with Flox-Exos, aKO-Exos (scale bar = 100 μm) (n = 4). (c, d) Representative muscle fiber immunofluorescent MyoD staining of CTX-induced C2C12 cells after transfection with miR-146a-5p mimics/inhibitors or co-treatment with Flox- Exos, aKO-Exos (scale bar = 100 μm) (n = 4). (e, f) Representative muscle fiber immunofluorescent Pax7 staining of CTX-induced C2C12 cells after transfec tion with miR-146a-5p mimics/inhibitors or co-treatment with Flox-Exos, aKO-Exos (scale bar = 100 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: In Vitro, Staining, Transfection

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 7 Adipose-derived miR-146a-5p is indispensable in muscle atrophy and repair. (a) RT-qPCR of miR-146a-5p in TA muscle cross-sections from injured (5, 7, and 15 days after CTX injection) Flox and aKO mice (n = 4). (b, c) Representative of MyHC immunofluorescent staining of TA muscle cross-sections from uninjured (Day 0) and injured (5, 7, and 15 days after CTX injection) Flox and aKO mice (scale bar = 100 μm) (n = 4). (d, e) Representative of MyoD immunofluorescent staining of TA muscle cross-sections from uninjured and injured Flox and aKO mice (scale bar = 100 μm) (n = 4). (f, g) Representative of Pax7 immunofluorescent staining of TA muscle cross-sections from both uninjured and injured Flox and aKO mice (scale bar = 100 μm) (n = 4). (h) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in TA muscle cross-sections from injured Flox and aKO mice (n = 4). (i) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos and Flox-Exos injection) aKO mice (n = 4). (j) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR measured by Western blot in TA muscle cross-sections from injured aKO mice (n = 3). (k) RT-qPCR of miR-146a-5p in TA muscle cross-sections from injured aKO mice treated with Exos (aKO-Exos and Flox-Exos injection) (n = 4). (l, m) Representative of MyHC immunofluorescent staining of TA muscle cross-sections from both uninjured and injured aKO mice treated with Exos (scale bar = 100 μm) (n = 4). (n, o) Representative of MyoD immunofluorescent staining of TA muscle cross-sections (scale bar = 100 μm) (n = 4). (p, q) Representative of Pax7 immunofluorescent staining of TA muscle cross-sections from both uninjured (Day 0) and injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox- Exos injection) aKO mice (scale bar = 100 μm) (n = 4). (r) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox-Exos injection) aKO mice (n = 4). (s) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos and Flox-Exos injection) aKO mice (n = 4). (t) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR measured by Western blot in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox-Exos injection) aKO mice (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Quantitative RT-PCR, Injection, Staining, Western Blot

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 8 miR-146a-5p prevents muscle atrophy by targeting IGF-1R. (a) miR-146a-5p has a target interaction with the 3’UTR of IGF-1R. (b) Relative luciferase activity was calculated by firefly luminescence/renilla luminescence (WT: pmiGLO- IGF-1R -WT, mut site 1 + 2: pmirGLO-IGF-1R -Mut1 + pmirGLO-IGF-1R- Mut2, mut site 1: pmirGLO- IGF-1R -Mut1, mut site 2: pmirGLO-IGF-1R -Mut2) (n = 10). (c) The expression of miR-146a-5p gene in C2C12 cells following transfection with siRNA-IGF-1R and co-treatment with siRNA-IGF-1R and miR-146a-5p inhibitor (n = 4). (d) CCK-8 result of C2C12 cells (n = 8). (e, f) EdU image and statistical analyses of C2C12 cells (scale bar = 100 μm) (n = 6). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in C2C12 cells (n = 4). (h, i) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results in C2C12 cells (n = 3). (j-l) Representative muscle fiber immunofluorescent MyHC staining, myotube diameter, and myotube fusion rate of C2C12 cells (scale bar = 100 μm) (n = 4). (m, n) Representative muscle fiber immunofluorescent MyoD staining and statistical results of C2C12 cells (scale bar = 100 μm) (n = 4). (o, p) Representa tive muscle fiber immunofluorescent Pax7 staining and statistical results of C2C12 cells (scale bar = 100 μm) (n = 4). (q) RT-qPCR analysis for IGF-1R, MyoD, MyoG, Pax7, Fbx32 and MuFR in C2C12 cells (n = 4). (r) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR by Western blot in C2C12 cells. (s) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 measured by Western blot in C2C12 cells. (n = 3). (t) Immunoprecipitation assay revealed an enrichment of P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 when introduced with IGF- 1R. (u) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in the TA muscles of Flox and aKO mice (n = 3). (v) RT-qPCR analysis for IGF-1R in C2C12 transfected with miR-146a-5p mimics/inhibitor (n = 6). (w) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 by Western blot in C2C12 transfected with miR-146a-5p mimics/inhibitor (n = 3). (x) RT-qPCR analysis for IGF-1R in aKO TA muscles injected aKO-Exos and Flox-Exos for 24 d (n = 3). (y) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in aKO TA muscles injected aKO-Exos and Flox-Exos for 24 d (n = 3). (z) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in TA muscle cross-sections from injured (5, 7 and 15 days after CTX injection) Flox and aKO mice (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Staining, Immunoprecipitation, Muscles, Injection

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 1. DDX4 forms cytoplasmic granules in cancer cells (A) Of the 88 known components of the CB, 22 have also been identified as CGAs (the dotted rectangular box, DDX4 highlighted in red). (B) Immunostaining of different human epithelial tissues with an anti-DDX4 antibody. DDX4 granules are absent from the normal epithelial tissues, but present (black arrows) in the cytoplasm of cancer cells in breast, colon, and lung adenocarcinoma; scale bar: 20 mm. Selected cancer cells are highlighted in the insets; scale bar: 10 mm. (C) Immunostaining of fibrosarcoma and leiomyosarcoma tissues as examples of DDX4+ cancers that are not of epithelial origin. Selected DDX4 granules are indicated with black arrows. Scale bar: 20 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Immunostaining

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 2. DDX4 granules are found in cancer-cell-line-derived xenograft tumors (A) Immunohistochemistry of UT-SCC-14-derived xenograft tumors with an anti-DDX4 antibody. Selected DDX4 granules are indicated with black arrows. Scale bar: 20 mm. Smaller rectangular box: no primary antibody control. (B) Immunofluorescence of UT-SCC-14 cultured cells and tumors with an anti-DDX4 antibody (red), nuclei are stained with DAPI (gray). DDX4 granules (white arrows) appear in xenograft tumor cells. Neg. Ctrl: no primary antibody. Scale bar: 10 mm. (C) Immunofluorescence of cultured PC3 cells and PC3-derived tumors with an anti-DDX4 antibody (red). DDX4 granules (white arrows) can be detected in tumors. Scale bar: 10 mm. (D) DDX4 IP from PC3 tumors and cells (3 biological replicates each) followed by western blotting. Rabbit IgG was used as a negative control. Graph: DDX4 signal was quantified, and the signal intensities were normalized to the IgG light-chain signal (asterisk) (p = 0.0274, Mann-Whitney U test, 2-tailed). (E) Immunofluorescence of PC3 spheroids with an anti-DDX4 antibody (red). Anti-a-tubulin antibody (green) visualizes cytoplasmic protrusions. White arrows point to selected DDX4 granules. Scale bar: 20 mm. (F) Immunofluorescence of DDX4 (red) in non-treated and puromycin-treated PC3 cells. DAPI (gray) stains the nuclei. Scale bar: 10 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Derivative Assay, Immunohistochemistry, Control, Cell Culture, Staining, Western Blot, Negative Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 3. DDX4 deletion compromises PC3 spheroid formation (A) The sequence of DDX4 gene after CRISPR-Cas9 in PC3 cells to visualize the deleted 103 nt. (B) Agarose gel of genomic PCR of WT and 2 DDX4-null PC3 cell clones (null1 and null2). (C) Proliferation curve of DDX4-null and WT PC3 cells. (D) Bar charts show the percentage of live cells, early apoptotic cells, and late apoptotic cells in WT and DDX4-null PC3 cells (3 replicates each). The chart at right shows only the early and late apoptotic cells. (E) Calcein AM-stained (green) WT and DDX4-null PC3 spheroids at days 5 and 10. Scale bar: 20 mm. (F) Area of spheroids formed by DDX4-null vs. WT PC3 spheroids (day 5: p = 0.000043, day 10: p = 0.000011, Mann-Whitney U test, 2-tailed). (G) Invasive processes (MaxApp in AMIDA) of DDX4-null vs. WT PC3 spheroids (day 5: p = 0.000011, day 10: p = 0.000022, Mann-Whitney U test, 2-tailed). (H) Immunofluorescence on WT and DDX4-null (number of replicates is 10 for each) PC3 spheroids with vimentin antibody (green). Nuclei were stained with DAPI (gray). Scale bar: 20 mm. (I) Calcein AM-stained (green) spheroids derived from WT and DDX4-null PC3 cells overexpressing either GFP or DDX4-GFP. Scale bar: 20 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Sequencing, CRISPR, Agarose Gel Electrophoresis, Clone Assay, Staining, MANN-WHITNEY, Derivative Assay

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 4. DDX4 deletion compromises PC3 tumor formation and growth (A) The volume of subcutaneous DDX4-null vs. WT PC3 xenograft tumors; 10 biological replicates each. The tumor progression was followed weekly for 4 weeks (Wk1: p = 0.000411, Wk2: p = 0.000119, Wk3: p = 0.00105, Wk4: p = 0.006798, Mann-Whitney U test, 2-tailed). (B) The weights of dissected DDX4-null vs. WT PC3 tumors (p = 0.0232, Mann-Whitney U test, 2-tailed). (C) Immunofluorescence with an anti-DDX4 antibody (red) validated the absence of DDX4 in DDX4-null PC3 tumor cells. Nuclei were stained with DAPI (gray). Scale bar: 20 mm. (D) Immunofluorescence on DDX4-null and WT PC3 tumors with an anti-vimentin antibody (green). Scale bar: 10 mm. (E) Western blotting of 3 biological replicates of WT and DDX4-null PC3 tumors with an anti-vimentin antibody. b-Actin was used as the loading control. The graph shows the quantification of vimentin signal (normalized to b-actin) (p = 0.0309, Mann-Whitney U test, 2-tailed). Data are represented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: MANN-WHITNEY, Staining, Western Blot, Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 5. Transcriptome is misregulated in DDX4-null PC3 cells and DDX4-null PC3 xenograft tumors (A) Hierarchical heatmaps show differentially expressed (|log2FC| R 1, adjusted p value %0.05) genes in DDX4-null vs. WT PC3 cells (left) and xenograft tumors (right). (B) Venn diagram shows the overlap between the genes upregulated in DDX4-null vs. WT PC3 xenograft tumors and cells. (C) Volcano plot shows the differential expression of tumor suppressor genes in DDX4-null vs. WT PC3 tumors, with significantly upregulated (green) and downregulated (orange) genes labeled. (D) Bar chart shows the log2FC (DDX4-null vs. WT tumors) of individually selected genes. (E) Bar chart shows the validation of the selected differentially expressed genes in DDX4-null vs. WT PC3 tumors (number of biological replicates is 3) by qRT-PCR (ADAM12: p = 0.0112, CDH6: p = 0.0161, CDH7: p = 0.0298, CEACAM1: p = 0.0468, SFRP1: p = 0.0293, and CLEC2B: p = 0.0105, Mann-Whitney U test, 2-tailed). (F) Western blotting image of CDH6, CDH1, and CDH2 protein expression in DDX4-null and WT PC3 tumors (3 biological replicates each). b-Actin was used as the loading control. Bar chart shows the quantification of the CDH1, CDH2, and CDH6 protein levels normalized by b-actin signal. CDH1: p = 0.0055, CDH6: p = 0.0004 (Mann-Whitney U test, 2-tailed, 3 biological replicates each). Data are represented as mean ± SEM. See also Figures S3 and S4 and Table S1.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Quantitative Proteomics, Labeling, Biomarker Discovery, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 6. DDX4 deletion affects the splicing landscape of cancer cells (A) IP of PC3 tumors (2 biological replicates) with an anti-DDX4 antibody, followed by western blotting with the same antibody. IgG IP was used as the negative control. IgG light and heavy chains are indicated with asterisks. (B) GO term enrichment analysis of the proteins interacting with DDX4 in PC3 xenograft tumors. Bubble plot visualizes the most enriched biological processes selected based on the adjusted p values. Gene ratio: The number of proteins that are associated with the GO term divided by the total number of proteins. Count: represents the number of proteins annotated to the GO term; the size of a bubble dot reflects the number of proteins. (C) Venn diagram illustrating 17 shared components (listed in the rectangular box) between the CB and the DDX4-interacting proteins.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Western Blot, Negative Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 7. DDX4 has prognostic significance in human cancers (A) Immunohistochemistry of normal head and neck squamous epithelium (HNSE, 3 representative examples) and HNSCC (bottom) with an anti-DDX4 antibody. The cells were counterstained with hematoxylin. HNSCC samples without granular DDX4 staining were scored as negative, while HNSCC samples with DDX4 granules (black arrows) were scored as positive. Scale bar: 20 mm. Survival curve shows 5-year survival (days) for DDX4 (n = 12/46) and DDX4+ (n = 34/46) patients (p = 0.008, log rank test). (B) Immunofluorescence on PC samples with an anti-DDX4 antibody. PC samples without granular DDX4 staining were scored as negative, while the PC samples with prominent DDX4 granules (white arrows) were scored as positive. Scale bar: 20 mm. Survival curve shows the progression-free survival (months) for DDX4

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Immunohistochemistry, Staining

Journal: Immunology letters

Article Title: Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling.

doi: 10.1016/j.imlet.2011.03.004

Figure Lengend Snippet: Fig. 3. TG2 null macrophages respond to LPS stimulation by an enhanced NF-B activation as compared to their wild-type counterparts, and this phenomenon is not related to an altered cell surface expression of CD14 or TLR4. (A) Flow cytometric analysis of cell surface CD14 (left) and TLR4 (right) expression of wild-type and TG2 null peritoneal macrophages. Open histograms on the left indicate isotype controls. (B) Quantitative RT-PCR analysis of TNF and IL6 mRNA expression in wild-type and TG2 null peritoneal macrophages cultured for 1 h with or without 100 ng/ml LPS. The results are representative of three independent experiments and are shown as mean ± SD. (C) Measurement of TNF mRNA stability in wild-type and TG2 null peritoneal macrophages. Cell were treated with 100 ng/ml LPS for 1 h followed by addition of 1 g/ml Actinomycin D. TNF mRNA was measured by quantitative RT-PCR. (D) Western blot analysis of IB degradation in wild-type and TG2 null macrophages after exposure to 100 ng/ml LPS. -actin was used as a loading control. (E) Determination of the amounts of nuclear p65 NF-B subunit in control and LPS-stimulated macrophages. Wild-type and TG2 null peritoneal macrophages were treated with 100 ng/ml LPS for the indicated time periods. Nuclear p65 subunit was detected with TransAM p65 kit. The results are representative of three independent experiments and are expressed as fold induction normalized to the wild-type control samples, and are shown as mean ± SD (*significantly different from wild-type, p < 0.05 determined unpaired Student’s t-test).

Article Snippet: Flow cytometry 5 × 105 peritoneal macrophages were labeled in 50 l PBS with FITC conjugated anti-CD14 antibody (Pharmingen) or rabbit-anti mouse TLR4 antibody (Santa Cruz Biotechnology) washed with PBS and incubated further with FITC-anti-rabbit antibody.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Control

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 1 NUAK1 co-immunoprecipitates with p53. Equal amount of cell extracts from A549 cells were immunoprecipitated with anti-NUAK1 antibody or normal rabbit IgG as negative control and were western blotted with anti-p53 antibody. Fifty percent of protein before immunoprecipitation was kept for input and was subjected to western blotting with anti-NUAK1 and anti- LKB1 antibodies. Vec, LKB1 and KDM indicate that A549 cells were stably transfected with vector control, wild-type (WT) LKB1 and kinase-deficient LKB1, respectively; L þ s and L þ c indicate that cells that stably expressed WT LKB1 were transiently transfected with NUAK1 siRNA pool or control siRNA as a control. Glucose: cells were incubated in medium without glucose for 2 h; glucose þ : no glucose starvation treatment.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 2 NUAK1 directly phosphorylates p53. (a) In vitro phosphorylation of p53 by E. coli-produced and LKB1-activated NUAK1. In kinase buffer that contained (g-32P) ATP, His-p53 was incubated with His-NUAK1 (N), or His-NUAK1 incubated with active- LKB1 and then isolated (NL) or His-NUAK1 incubated with heat-inactivated active-LKB1 and then isolated (NiL). After separation by SDS–PAGE, proteins were transferred to PVDF membrane and detected by autoradiography. His-p53 and His-NUAK1 were also detected by anti–p53 and anti-NUAK1 antibodies. (b) In vitro phosphorylation of p53 by E. coli-produced mutants of NUAK1. His-p53 was incubated with His-NUAK1 (N), His-NUAK1 (T211E) (TE) or His-NUAK1 (T211D) (TD), and then detected by autoradiography. His-p53, His-NUAK1 and mutants were also examined by western blotting. (c) In vitro phosphorylation of p53 by NUAK1 and mutants produced in HEK293T cells. His-p53 was incubated with His-NUAK1 (N), T211A mutant (TA) or kinase-dead mutant K84A (KA) and then detected by autoradiography. Western blotting was the same as in (b). (d) Phosphorylation of p53 in Hep3B cells. Hep3B cells were stably transfected with vector control (Vec) or WT p53 (p53) or p53 and transiently expressed NUAK1 (p þ N) or p53 and NUAK1 with ATM siRNA pool (pNs) or p53 and NUAK1 with control siRNA (pNc). After incubated in glucose þ or glucose medium for 2 h, cells were lysed and subjected to western blotting with phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53, anti-NUAK1, anti-ATM and b-actin antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vitro, Phospho-proteomics, Produced, Incubation, Isolation, SDS Page, Membrane, Autoradiography, Western Blot, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Control

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 3 LKB1 activation of NUAK1 stimulates phosphorylation of p53. (a) LKB1-dependent p53 phosphorylation requires NUAK1. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) or WT LKB1 and transiently transfected with control siRNA (L þ c). Cells were incubated in glucose þ or glucose medium for 2 h. Western blotting was done using phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53 antibody, anti-NUAK1 antibody, anti-LKB1 antibody and b-actin antibody. (b) Requirement of NUAK1 kinase activity in LKB1-dependent p53 phosphorylation. A549 cells stably transfected with LKB1 ( þ ) or vector control () were transiently transfected with NUAK1 ( þ ), T211A (TA), kinase-dead mutant K84A (KA) or vector control (Vec), and treated under glucose starvation for 2 h. Cells were lysed and western blotting was performed as in (a). (c) In vivo phosphorylation assay of NUAK1 by LKB1. A549 cells that stably expressed WT LKB1 , vector control (Vec) or kinase-deficient LKB1 (KDM) were transiently transfected with WT NUAK1 or NUAK1 T211A mutation. Transfection was the same in (d), (e) and (f). The cells were subjected to glucose starvation for 2 h and incubated for 3 h with [32P] Pi (300 cpm/pmol; Furi). Cells were then lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The immunoprecipitates were separated by SDS–PAGE and subjected to autoradiography. (d) Cells were treated with 200 mM AMP. (e) In vitro kinase assay of NUAK1. After being subjected to glucose starvation for 2 h, cells were lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The in vitro kinase activity of immunoprecipitates was assayed by measuring the 32P labeling of SAMS peptide. One unit of activity was defined as 1 nmol SAMS peptide phosphorylated per minute. (f) Cells were treated with 200 mM AMP.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Activity Assay, Mutagenesis, In Vivo, Immunoprecipitation, SDS Page, Autoradiography, In Vitro, Kinase Assay, Labeling

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 4 Cell cycle arrest induced by LKB1/NUAK1 requires p53. (a) A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 ( þ ). Cells stably expressed WT LKB1 were also transiently transfected with ( þ ) or without () WT NUAK1, NUAK1 siRNA pool (siRNA) or control siRNA (Ctl-si). After synchronization, cells were treated with glucose medium. Cells were then harvested, stained with propidium iodide and analyzed by flow cytometry. Each analysis was carried out in triplicate and also in (b). (b) A549 cells were stably transfected with vector control (Vec) or WT LKB1 ( þ ), and transiently transfected with p53 ( þ ), vector control (Vec), p53 S15A mutant (S15A), p53 S392A mutant (S392A), p53 siRNA pool (siRNA) or control siRNA (Ctl-si). Cells were treated as in (a) and subjected to flow cytometry analysis.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Staining, Cytometry, Mutagenesis

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 5 p21/WAF1 transcriptional activity induced by LKB1/ NUAK1. (a) Quantitative RT–PCR analysis of p21/WAF1 transcription. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM), WT LKB1 (LKB1) and transiently transfected with NUAK1 siRNA pool (L þ s), or WT LKB1 and transiently transfected with control siRNA (L þ c). RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA and displayed as fold change relative to the value obtained from cells transfected with vector control, which was set at 1. (b) Western blotting of endogenous p21 protein. Transfection was the same as in (a).

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 6 NUAK1 interacts with p53 in the nucleus and binds to p21/WAF1 promoter. (a) Endogenous NUAK1 was present in the p53RE region of p21/WAF1 promoter. A549 cells were stably transfected with vector control (Vec), wild-type LKB1 (LKB1), kinase- deficient LKB1 (KDM), or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s). Cells were subjected to glucose starvation for 2 h. ChIP was done using anti-NUAK1 antibody and normal rabbit IgG was used as a negative control for the specificity of the immunoprecipitation. Cells transiently transfected with NUAK1 siRNA pool were included as a negative control. Quantitative PCR was done with the specific primers for p53RE of p21/WAF1 promoter and the bars represent normalized quantitative PCR values expressed as percentage of input. (b) ChIP assay of NUAK1 at p21/WAF1 TATA-50UTR region. (c) ChIP assay of p53 at p21/WAF1 promoter p53RE region. (d) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region. (e) The binding of NUAK1 to p53RE required wild-type p53. p53-null Hep3B cells were stably transfected with wild-type p53 (p53), p53 S15A mutant (S15A), p53 S392A mutant (S392A) or vector control (Vec). ChIP assay was done as described in A and p53-expressing cells transiently transfected with NUAK1 siRNA pool were included as a negative control (p þ s). (f) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region in Hep3B cells. p53-expressing cells transiently transfected with LKB1 siRNA pool were included as a negative control (p þ s). (g) Co-immunoprecipitation analysis of endogenous NUAK1 and p53 from A549 cells that stably expressed vector control (Vec), wild-type LKB1 (LKB1), kinase-deficient LKB1 (KDM) or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) as control. Cells were treated with glucose– medium and then fractionated (N: nuclear fraction; C: cytoplasmic fraction). Equal amounts of protein from each sample were immunoprecipitated using anti-NUAK1 antibody or using normal rabbit IgG as a negative control. Fifty percent of protein before immunoprecipitation was kept for input, and was subjected to western blotting with anti-NUAK1 and anti-p53 antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Expressing, Western Blot

Journal: Alcoholism, clinical and experimental research

Article Title: Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

doi: 10.1111/acer.12044

Figure Lengend Snippet: Fig. 1. Ethanol (EtOH) induces the expression of nicotinic acetylcholine receptors (nAChRs) in lung fibroblasts. (A) Upper panel: RT-PCR analysis of primary lung fibroblasts (4 9 104 cells/well) in 12-well plates treated with or without EtOH for 24 hours. Afterward, cells were washed, harvested, and processed for RT-PCR analysis of nAChR mRNA. PCR products were analyzed on 1% agarose gel stained with ethidium bromide. Lower panel: Quantifi- cation of nAChR mRNA using real-time RT-PCR analysis of cells using a Cepheid Smart Cycler. mRNA values were normalized to 18S and shown as means SD. Note that a4 and a9 nAChR mRNA levels were significantly increased in lung fibroblasts treated with EtOH. *Significant difference from nontreated cells (n = 4; p < 0.01). (B) Upper panel: Primary lung fibroblasts (1 9 106 cells/ml) in 6-well plates treated with or without EtOH for 24 hours followed by Western blot analysis for a4, a9, a10, or b2 nAChR protein expression. Duplicate blots were analyzed for actin expression and used as loading controls. Lower panel: Quantification of protein levels using a Bio-Rad GS-800 laser densitometer. Note that only a4 nAChR protein levels were signifi- cantly elevated in fibroblasts treated with EtOH. *Significant difference from nontreated cells (n = 4; p < 0.01).

Article Snippet: Blots were washed, incubated with primary antibody against fibronectin (1:1,000 dilution; Sigma) or a4, a9, a10, b2 nAChR (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 24 hours at 4°C, washed and incubated with secondary goat anti-rabbit IgG (horse radish peroxide- ETHANOL SENSORS IN LUNG FIBROBLASTS 915 15300277, 2013, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/acer.12044 by U niversity O f T he Philippines, W iley O nline L ibrary on [08/02/2024].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Western Blot

Journal: Alcoholism, clinical and experimental research

Article Title: Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

doi: 10.1111/acer.12044

Figure Lengend Snippet: Fig. 2. Acetylcholine mimics the effect of ethanol (EtOH); dihydro-b-erythroidin hydrobromide (DbH), an a4 nicotinic acetylcholine receptor (nAChR) inhibitor, blocks EtOH-induced fibronectin expression. (A) Acetylcholine mimics the effect of EtOH on the induction of fibronectin promoter expression. Primary mouse lung fibroblasts isolated from C57BL/6 mice transgenic for the fibronectin–luciferase promoter (4 9 104 cells/well) were added to 48-well plates and cultured in the presence of varying concentrations of acetylcholine (100 to 500 lM) at 37°C for 24 to 96 hours, harvested, and cell extracts were processed for induction of fibronectin promoter expression by luciferase assay. Quantification was performed using a Labsystems Luminoskan Ascent Plate Luminometer, results were recorded as relative luciferase units, and all samples were normalized for total protein as determined by the Bradford method. *Significant difference from control nontreated cells (n = 8; p < 0.01). (B) Induction of fibronectin promoter activity by EtOH is inhibited by pretreatment with an a7 nAChR inhibitor, a-bungarotoxin (a-BGT) and a competitive inhibitor of a4 nAChR, DbH. Primary mouse lung fibroblasts iso- lated from C57BL/6 mice transgenic for the fibronectin–luciferase promoter (1 9 104 cells/well) were added to 48-well plates and cultured in the presence of physiological concentrations of EtOH (60 mM) at 37°C for 24 hours, harvested, and cell extracts were processed for induction of fibronectin promoter expression by luciferase assay. Quantification was performed using a Labsystems Luminoskan Ascent Plate Luminometer, results were recorded as rela- tive luciferase units and all samples were normalized for total protein as determined by the Bradford method. Both a-BGT and DbH were able to abrogate the fibronectin promoter induction by EtOH. *Significant difference from EtOH-treated cells (n = 8; p < 0.01). (C) DbH inhibits EtOH-induced fibroblast proliferation. Primary mouse lung fibroblasts (1 9 104 cells/ml) were added to 96-well plates, treated with or without EtOH in the presence or absence of the a4 nAChR inhibitor DbH for 24 to 72 hours. Afterward, cell proliferation was measured by the Cell Titer-Glo Luminescent Cell Viability. Quantification was performed using a Labsystems Luminoskan Ascent Plate Luminometer, results were recorded as relative luciferase units. Note that DbH significantly inhibited the increase in cell proliferation at 72 hours of EtOH treatment. *Significant difference from control nontreated cells at 72 hours (n = 8; p < 0.01). **Significant difference from EtOH-treated cells at 72 hours (n = 8; p < 0.01).

Article Snippet: Blots were washed, incubated with primary antibody against fibronectin (1:1,000 dilution; Sigma) or a4, a9, a10, b2 nAChR (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 24 hours at 4°C, washed and incubated with secondary goat anti-rabbit IgG (horse radish peroxide- ETHANOL SENSORS IN LUNG FIBROBLASTS 915 15300277, 2013, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/acer.12044 by U niversity O f T he Philippines, W iley O nline L ibrary on [08/02/2024].

Techniques: Expressing, Isolation, Transgenic Assay, Luciferase, Cell Culture, Control, Activity Assay

Journal: Alcoholism, clinical and experimental research

Article Title: Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

doi: 10.1111/acer.12044

Figure Lengend Snippet: Fig. 3. a4 and a9 nicotinic acetylcholine receptor (nAChR) siRNAs inhibit ethanol (EtOH)-induced fibronectin expression. (A) Western blot and mRNA analysis of the effect of a4, a9, a10, or b2 nAChR or control nontarget siRNA knockdown in transfected primary lung fibroblasts. Upper panel shows the ability of the siRNA to eliminate the expression of the nAChRs, while the lower panel demonstrates the ability of the siRNA to down-regulate mRNA expression. Western blot gels were stripped and reprobed for GAPDH expression to control for loading. The 18S subunit was used to normalize mRNA expression for RT-PCR analysis. (B) Induction of fibronectin mRNA expression by EtOH is inhibited by knock down of both the a4 and a9 nAChR by siRNA. Primary mouse lung fibroblasts (4 9 104 cells/well) transfected with a4, a9, a10, or b2 nAChR or control nontarget siRNA in 12-well plates for 24 hours were treated with or without EtOH for an additional 24 hours. Afterward, cells were washed, harvested, and processed for RT-PCR analysis of fibronectin mRNA. Relative fibronectin mRNA values were normalized to 18S and shown as means SD. Note that a4 and a9 nAChR siRNA blocked the EtOH induced fibronectin mRNA expression when compared to control cells or cells transfected with control nontarget siRNA. Both a10 and b2 nAChR siRNA failed to block the effects of EtOH. *Significant difference from control or nontarget siRNA-treated cells (n = 4; p < 0.01). (C) Induction of fibronectin protein expression by EtOH is inhibited by knock down of a4 and a9 nAChR by siRNA. Primary mouse lung fibroblasts (4 9 104 cells/well) transfected with a4, a9, a10, or b2 nAChR or control nontarget siRNA in 12-well plates for 24 hours were treated with or without EtOH for an additional 24 hours. Afterward, cells were washed, harvested, and processed for Western blot analysis of fibronectin. Identical blots were incubated for b-actin expression and used for gel-loading control. Bars in graph are shown as means SD. Note that a4 and a9 nAChR siRNA blocked the EtOH-induced fibronectin protein expression when compared to control cells or cells transfected with control nontarget siRNA. Both a10 and b2 nAChR siRNA failed to block the effects of EtOH. NS, control nontarget siRNA; FN, fibronectin.

Article Snippet: Blots were washed, incubated with primary antibody against fibronectin (1:1,000 dilution; Sigma) or a4, a9, a10, b2 nAChR (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 24 hours at 4°C, washed and incubated with secondary goat anti-rabbit IgG (horse radish peroxide- ETHANOL SENSORS IN LUNG FIBROBLASTS 915 15300277, 2013, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/acer.12044 by U niversity O f T he Philippines, W iley O nline L ibrary on [08/02/2024].

Techniques: Expressing, Western Blot, Control, Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Incubation

Journal: Alcoholism, clinical and experimental research

Article Title: Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

doi: 10.1111/acer.12044

Figure Lengend Snippet: Fig. 4. Ethanol (EtOH) stimulates, while knock-down of a4 nicotinic ace- tylcholine receptor (nAChR) blocks the binding of a-bungarotoxin (a-BGT). Primary lung fibroblasts (1 9 105 cells/ml) transfected with or without con- trol nontarget or a4 shRNA were plated into 24-well plates. Cells were then treated with or without EtOH for 24 hours. Some samples were pretreated with nicotine (2 mM) for 1 hour prior to the addition of 5 nM CF488A- labeled a-BGT (Biotium). Cells were incubated an additional 3 hours at 37°C and 5% CO2. Afterward, the cells were washed twice for 5 minutes with binding media, washed once for 15 minutes with TBS (10 mM Tris– HCl, pH 8.0, 150 mM NaCl), and washed once for 5 minutes with PBS. CF488A-labeled a-BGT bound to surface nAChRs was quantified using a Beckman Coulter AD-340 spectrophotometer. Values were normalized to total protein and shown as means SD. Nicotine, used as control, inhib- ited a-BGT. *Significant difference from control cells (n = 4; p < 0.01). **Significant difference from EtOH-treated control cells at 72 hours (n = 8; p < 0.01). Csh, primary lung fibroblasts transfected with control nontarget shRNA; a4sh, primary lung fibroblasts transfected with a4 shRNA. Nic, nic- otine.

Article Snippet: Blots were washed, incubated with primary antibody against fibronectin (1:1,000 dilution; Sigma) or a4, a9, a10, b2 nAChR (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 24 hours at 4°C, washed and incubated with secondary goat anti-rabbit IgG (horse radish peroxide- ETHANOL SENSORS IN LUNG FIBROBLASTS 915 15300277, 2013, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/acer.12044 by U niversity O f T he Philippines, W iley O nline L ibrary on [08/02/2024].

Techniques: Knockdown, Binding Assay, Transfection, shRNA, Labeling, Incubation, Spectrophotometry, Control, Inhibition

Journal: Alcoholism, clinical and experimental research

Article Title: Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

doi: 10.1111/acer.12044

Figure Lengend Snippet: Fig. 6. Ethanol (EtOH) increases a4 nicotinic acetylcholine receptor (nAChR) expression in vivo. (A) Immunohistochemical analysis of a4 nAChR expression in the lungs of animals treated with or without EtOH. The lungs from control animals showed little staining for a4 nAChR, while the lungs of EtOH-treated animals exhibited a dramatic increase in staining for a4 nAChR in periairway cells, peribronchial tissue, primary lung fibroblasts, vascular structures, within the interstitium, and alveolar macrophages. (B) RT-PCR analysis of a4 nAChR mRNA expression in lung of both mice and rats treated with or without EtOH. Upper panel: RT-PCR analysis of nAChR mRNA, PCR products were analyzed on 1% agarose gel stained with ethidium bromide. Lower panel: Quantification of nAChR mRNA using a Bio-Rad GS-800 laser densitometer. mRNA values were normalized to actin and shown as means SD. Note that a4 mRNA levels were significantly increased in the lungs of animals treated EtOH. *Significant difference from nontreated ani- mals (n = 4; p < 0.01). (C) Western blot analysis of a4 nAChR protein expression in the lung of both mice and rats treated with or without EtOH.

Article Snippet: Blots were washed, incubated with primary antibody against fibronectin (1:1,000 dilution; Sigma) or a4, a9, a10, b2 nAChR (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 24 hours at 4°C, washed and incubated with secondary goat anti-rabbit IgG (horse radish peroxide- ETHANOL SENSORS IN LUNG FIBROBLASTS 915 15300277, 2013, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/acer.12044 by U niversity O f T he Philippines, W iley O nline L ibrary on [08/02/2024].

Techniques: Expressing, In Vivo, Immunohistochemical staining, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot