Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Functional effects of HOTAIR inhibition on HeLa cells. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h. HOTAIR knockdown efficiency was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. B, Apoptosis was determined by annexin V staining and flow cytometry. C, Apoptosis rate of HeLa cells at 48 h after transfection with siHOTAIR or siNC. D, Effect of HOTAIR knockdown on cell cycle progress. The percentage of cells in the G1 phase was significantly decreased whereas that in S phase and G2/M phase was increased after HOTAIR inhibition. E, HOTAIR knockdown in HeLa cells significantly inhibits cell growth. F, Effect of HOTAIR knockdown on cell invasion, as determined in a Boyden chamber assay. G, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. H, HOTAIR inhibition led to a significant reduction of cell migration as determined by a wound-healing assay. I, Quantification of the wound healing assay. Data are presented as means ± S.D. and represent results from three independent experiments. Statistically significant differences are indicated: *p < 0.05; **p < 0.01.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Functional Assay, Inhibition, Transfection, Knockdown, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Boyden Chamber Assay, Migration, Wound Healing Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Quantitative proteomic identification of HOTAIR-regulated proteins in HeLa cells. A, Workflow for the identification of HOTAIR-regulated proteins. HeLa cells were differentially labeled by growing them in medium containing light or heavy amino acids (SILAC). Proteins were extracted from the labeled cells 48 h after transfection with siHOTAIR or siNC, then equal amounts of protein from each sample were combined. The protein mix was separated by 12% SDS-PAGE and the resulting gel was cut into 30 sections. Each of the fractions was in-gel digested and analyzed via LC-MS/MS. B, Heatmap showing the expression of differentially expressed proteins after HOTAIR inhibition. C, PANTHER Protein Class ontology classification of the 170 proteins differentially expressed after HOTAIR expression silencing. HOTAIR-regulated proteins were classified into 22 classes.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Labeling, Multiplex sample analysis, Transfection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: VIM contributes to the effects of HOTAIR knockdown. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h and VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. B, HeLa cells were transfected with siVIM for 48 h, then VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. C, Western blot analysis of VIM protein expression 48 h after transfection with siVIM (siVIM-I, siVIM-II and siVIM-III) or siNC. GAPDH was used as an internal control. D, HeLa cells were transfected with pVIM or pEGFP. VIM expression levels were determined by Western blotting at 24 h and 48 h after transfection. EGFP serves as the negative control and GAPDH as the loading control. E, Effect of VIM knockdown and overexpression on cell invasion as determined with a wound-healing assay. F, Quantification of the wound healing assay. G, Effect of VIM knockdown and overexpression on cell invasion, as determined with a Boyden chamber assay. H, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *p < 0.05; **p < 0.01. I, HOTAIR suppressed tumor growth and regulated VIM expression in nude mice. HeLa cells transfected with siHOTAIR or siNC and HeLa cells expressing either control shRNA or shHOTAIR were injected subcutaneously into the right flank of nude mice. After 20 days, mice were sacrificed and tumors were dissected and weighed. Representative photographs of xenografts were taken 20 days after injection of HeLa cells transfected with siHOTAIR or HeLa-KD cells. J, Quantification of tumor weight. Data are presented as means ± S.D. (n = 5). K, Western blotting of VIM protein expression in tumors excised from the mice indicated 20 days after injection.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Over Expression, Wound Healing Assay, Boyden Chamber Assay, shRNA, Injection

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Inhibition of HOTAIR or overexpression of vimentin affects organization of the vimentin IF network. A, Representative confocal microscopy images showing the organization of vimentin IF in HeLa cells after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells B, Quantification of vimentin IF collapse in cells in A. C, Representative cells showing organization of vimentin IF under super-resolution microscopy after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells. D, Quantification of the diameter of vimentin IFs in the cells in C. (Scale bars: 30 μm in A, 5 μm in C). At least three independent experiments were performed under each condition with at least 100 cells (B) or five 15 × 15 μm images of different cells (D) quantified per experiment. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Inhibition, Over Expression, Confocal Microscopy, Knockdown, Super-Resolution Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Inhibition of HOTAIR leads to mitochondrial dysfunction. A, The concentration of UQCR in HeLa cells decreased after HOTAIR knockdown as measured using ELISA assays. B, Effect of HOTAIR knockdown on cellular ROS production as detected by flow cytometry analysis. C, Quantification of DCF fluorescence in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. D, Images of cellular glucose photographed under a confocal microscope. E, HOTAIR affects glucose uptake in HeLa cells. Cells had lower cellular glucose levels after HOTAIR knockdown. Glucose uptake was measured by FACS, following 0.5 h exposure to 2-NBDG (100 μm). F, Quantification of the fluorescence of cellular glucose in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. G, Electron microscopy images of (a) siNC cells and (b) siHOTAIR cells. Labels: M = mitochondria, n = nucleus. H, HOTAIR knockdown affects the mitochondrial membrane potential. Mitochondria were stained with MitoTracker Deep Red and representative images were obtained by confocal microscopy. I, HeLa cells were stained against JC-1 for flow cytometry after HOTAIR knockdown. There was a significant increase in the number of cells with green fluorescence (FL1 (R3)), indicating a decrease in the Δψ. J, The mean fluorescence intensity ratio (FL2/FL1) in HeLa cells after HOTAIR knockdown. CCCP was the positive control. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Inhibition, Concentration Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Microscopy, Electron Microscopy, Membrane, Staining, Confocal Microscopy, Positive Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Proposed model depicting the molecular mechanism of HOTAIR in regulating migration and invasion of HeLa cells. HOTAIR promotes cell migration and invasion in HeLa cells via different mechanisms. HOTAIR may serve as a molecular scaffold linking two distinct histone modification complexes to regulate hundreds of genes (12). HOTAIR regulates the expression and organization of vimentin. Our functional study demonstrated that vimentin contributes to the decreased migration and invasion capability of HeLa cells caused by inhibition of HOTAIR. The mitochondrial dysfunction caused by inhibition of HOTAIR may be another cause of the decreased migration and invasion capability of HeLa cells. The combination of all these mechanisms regulates the expression of hundreds of proteins and promotes cell migration and invasion. Vimentin may be a key molecule in HOTAIR-mediated oncogenic signaling.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Migration, Modification, Expressing, Functional Assay, Inhibition

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Schematic illustration of loop-mediated isothermal amplification (LAMP). (B) Schematic illustration of CRISPR-Cas12a detection. Step 1: LAMP products are obtained. Protospacer adjacent motif (PAM) sites guide the CRISPR/Cas12a-gRNA complex to recognize target sites. Step 2: Cas12a effectors are activated. Step 3: The activated effectors nonspecifically cleave single-stranded DNA reporter molecules by trans-cleavage. (C) Schematic illustration of the LAMP-CRISPR/Cas12a assay workflow. The LAMP-CRISPR/Cas12a assay involves three closely linked steps: rapid template preparation (step 1), LAMP reaction (step 2), and CRISPR-Cas12a cleavage and signal detection (step 3).

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: Amplification, CRISPR

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Fluorescence changes of three crRNA reactions for 1 h detected by CRISPR-Cas12a fluorescence assays. Values are shown in the graph as means ± SD (n = 3). (B) Fluorescence curves at different reaction temperatures. (C) Fluorescence curves of different crRNA concentrations. (D) Effects of different LAMP reaction times on the Cas12a reaction. NTC: no template control.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: Fluorescence, CRISPR

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Specificity of the CRISPR-Cas12a enhanced fluorescent assay evaluated by its ability to distinguish base mismatches. Protospacer adjacent motif (PAM) sequences are shown in red; base mismatches are shown in green.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Fluorescence

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Specificity of the LAMP-CRISPR/Cas12a assay evaluated by its ability to distinguish B xylophilus strains and related species. B.C: Botrytis cinerea ; B.M: B mucronatus ; QY: B xylophilus (from Liaoning); NJ: B xylophilus (from Nanjing); CQ: B xylophilus (from Chongqing); HS: B xylophilus (from Anhui); B.D: B doui ; NTC: negative control (ddH2O). (B) End-point fluorescence visualization of the specificity test. (C) Detection of base changes or deletions in target sequences by LAMP-CRISPR/Cas12a assay by measuring fluorescence intensity under blue-light irradiation 1 h after the reaction. Panel 1: From left to right, 0–5 base changes; the last tube is the negative control. At four base changes, the fluorescence intensity dropped significantly. Panel 2: From left to right, 0–5 base deletions. the last tube is the negative control. At five base deletions, the fluorescence intensity dropped significantly.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Negative Control, Fluorescence, Irradiation

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: (A) Sensitivity of the LAMP-CRISPR/Cas12a assay for B xylophilus detection. Purified B xylophilus genomic DNA was diluted by 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 , 10 −7 , and 10 −8 to give DNA concentrations of 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, 6.64 pg/µL, 0.664 pg/µL, 66.4 fg/µL, and 6.64 fg/µL. Different concentrations of DNA were detected by LAMP-CRISPR/Cas12a assay and visualized by blue light. (B) Results of the LAMP-CRISPR/Cas12a assay visualized by lateral flow biosensor. (C) Real-time fluorescence profiles of DNA at different concentrations in LAMP-CRISPR/Cas12a assay. (D) Sensitivity of the PCR assay for B xylophilus detection. The image shows amplification bands targeting the SYG-2 gene. Lanes 1–6 lanes are B xylophilus genomic DNA dilutions 664 ng/μL, 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, and 6.64 pg/µL (3 replicates per concentration). (E, F) Different concentrations of DNA were detected by traditional LAMP and visualized by HNB and SYBRgreenI. (G) Real-time fluorescence curves of DNA with different concentrations in LAMP.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, Purification, Fluorescence, Amplification, Concentration Assay

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Comparison of the LAMP-CRISPR/Cas12a Assay with Traditional LAMP for detection of B. xylophilus .

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques:

Journal: Frontiers in Plant Science

Article Title: Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

doi: 10.3389/fpls.2022.1075838

Figure Lengend Snippet: Processing scheme for the LAMP-CRISPR/Cas12a assay includes the following steps: DNA extraction, isothermal amplification reactions, and CRISPR-Cas12 detection of the B xylophilus by fluorescence.

Article Snippet: For lateral flow detection, 100 µL HybriDetect Assay Buffer and 10 μl aliquot of products from the CRISPR-Cas12a trans-cleavage mixture (250 nM Cas12a, 500 nM crRNA, 400 nM ssDNA-lateral flow biosensor reporter, 2 µL LAMP product, and 2.5 µL NEBuffer 2.1) were added to a reaction tube.

Techniques: CRISPR, DNA Extraction, Amplification, Fluorescence

Journal: Cell Death & Disease

Article Title: TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons

doi: 10.1038/s41419-018-0369-4

Figure Lengend Snippet: a Representative recording of population spikes (each purple vertical line), and bursts (purple clusters with >4 spikes/s) from an individual electrode of a multichannel electrode array. Representative tracings show averages of spikes/second/electrode, and associated scatter plots show quantitation of spike and burst rates for ( b – e ) Control, ( f – i ) ADEV-IL-1β (particle dose of 50 ADEVs/cell), ( j – m ) ADEV-IL-1β+ Scrambled oligonucleotide (Scr In, 20 pmole), and ( n – q ) ADEV-IL-1β+ oligonucleotide inhibitors for miR-125 and miR-16 (Combined In, 20 pmole each). Data are mean ± SEM. Paired t -tests were performed to compare spike and burst rate of each electrode before and after treatment. *** p < 0.001 increased compared to baseline and ### p < 0.001 decreased compared to baseline

Article Snippet: In brief, hippocampal tissues were separately dissociated by gentle trituration in a calcium-free Hank’s balanced salt solution and centrifuged at 1000× g . Cells were resuspended in Neurobasal media (Gibco) containing B27 supplement (Thermo Fisher Scientific), 1% antibiotic/antimitotic solution (104 Unit of penicillin G/ml, 10 mg streptomycin/ml and 25 μg amphotericin B/ml) (Sigma), and plated at a density of 40,000 cells/ml in 96-well plates (Corning) coated with polyethyleneimine (Sigma) or 160,000 cells/ml in multichannel electrode array chambers (Harvard Apparatus) coated with polyethyleneimine (Sigma) and laminin (Sigma).

Techniques: Quantitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: MAMs formation boosted in mice fed with HFHC diets and PA-stimulation hepatocytes. A Transmission electron microscopy (TEM) images of hepatic tissue from C57BL/6 J mice fed a regular chow diet or a high-fat, high-carbohydrate (HFHC) diet for 16 weeks, showing the morphology of MAMs (× 60,000, scale bar 600 nm; n = 4 mice per group). B Confocal microscopy images displaying the colocalization of the ER and mitochondria in AML12 and HepG2 cells treated with BSA or palmitic acid (PA). Colocalization is indicated by the merged fluorescence of DsRed2-ER-5 (red) and Mito-Tracker Green. The Pearson’s coefficient graph quantifies the degree of colocalization (× 1200, scale bar 5 μm; n = 4 per group). C - D Western blot analysis of MAMs-related Ca 2 ⁺ channel proteins, including IP3R1, GRP75, and VDAC1, in homogenates and MAMs fractions from the liver of NAFLD mice and PA-stimulated AML12 cells. β-tubulin or calreticulin was used as a loading control. Data are presented as means ± SD. * P < 0.05, ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s. indicates no significant difference

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Transmission Assay, Electron Microscopy, Micro-arrays for Mass Spectrometry, Confocal Microscopy, Fluorescence, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: SIRT1 overexpression inhibited the Mito-ER contact, Ca 2+ dyshomeostasis, and mitochondria dysfunction induced by PA stimulation in hepatocytes. A Confocal microscopy images displayed the colocalization of ER and mitochondria in AML12 cells with stable overexpression of SIRT1 under BSA or PA exposure for 24 h. The colocalization is indicated by the merge of DsRed2-ER-5 and Mito-Tracker Green; and the Pearson’s coefficient graph quantifies the colocalization (× 1200, Scar bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1), β-tubulin served as a loading control. C Measurement of Ca 2 ⁺ dynamics. The left panels show the time course of the D1ER ratio and pCAG-Mito-Gcamp5G. The fluorescence was recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak or delta troughs are shown in the right panels ( n = 3 per group). D DCFH-DA/DAPI staining for analyzing the production of ROS (× 1200, Scar bar, 5 μm, n = 4 per group). E Flow cytometry analysis of mitochondrial superoxide levels ( n = 3 per group). F: BODIPY 493/503 staining for analyzing the mitochondrial membrane potential (× 200, Scar bar, 50 μm; n = 4 per group). Data were presented as the means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Over Expression, Confocal Microscopy, Western Blot, Expressing, Control, Fluorescence, Injection, Staining, Flow Cytometry, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: SIRT1 overexpression relieved ROS overgeneration and mitochondria dysfunction through the reduction of MAMs-mediated mitochondria Ca 2+ overload. A Confocal microscopy images displayed the colocalization of ER and mitochondria in AML12 cells with stable overexpression of SIRT1 transfected with Mock or Mito-ER linker vectors. The merge of ER Tracker Blue-White and Mito-Tracker Green indicates the colocalization. The Pearson’s coefficient graph quantifies the colocalization (× 1200, Scar bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1), with β-Tubulin serving as a loading control. C Mitochondria Ca 2 ⁺ levels. The left panels show the time course of the pCAG-Mito-Gcamp5G. The fluorescence was recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak are shown in the right panels ( n = 3 per group). D Flow cytometry analysis of ROS levels ( n = 3 per group). Data were presented as the means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Over Expression, Confocal Microscopy, Transfection, Western Blot, Expressing, Control, Fluorescence, Injection, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Activated SIRT1 blocked the aberrant formation of MAMs through the distribution of the interaction of MDM2 with MAMs-related Ca 2+ channels protein. A Differential gene expression of MAMs-related genes was analyzed by RNA-Seq from SIRT1-NC + PA vs. SIRT1-NC and SIRT1-NC + PA vs. SIRT1-OE + PA. Gray dots: Nonsignificant; Red dots: Significantly upregulated; Blue dots: Significantly downregulated; Black dots: MAMs-related genes. B Western blot analysis of MDM2 expression in MAMs fractions from the liver tissue of NAFLD mice treated with SRT1720 and PA-stimulated AML12 cells overexpressing SIRT1. Calreticulin served as a loading control. C Western blot analysis of MDM2 in whole-cell homogenates from the liver tissue of NAFLD mice treated with SRT1720 and PA-stimulated AML12 cells overexpressing SIRT1. β-Tubulin served as a loading control. D Interaction of MDM2 and MAMs-related Ca 2 ⁺ channels was analyzed by Co-IP in PA-stimulated AML12 cells overexpressing SIRT1 and in NAFLD mice treated with SRT1720. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Gene Expression, RNA Sequencing, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Inhibition of MDM2 expression reduced the MAMs formation enrichment caused by PA-stimulation and Mito-ER linker transfected in hepatocytes. A Confocal microscopy images showing the colocalization of ER and mitochondria in AML12 cells transfected with MDM2 constructs under BSA or PA exposure for 24 h. Colocalization is indicated by the merge of DsRed2-ER-5 and Mito-Tracker Green, and Pearson’s coefficient graph quantifies the colocalization (× 1200; scale bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1) in homogenates and MAMs fractions from AML12 cells. β-tubulin served as a loading control. C Co-IP analysis for IP3R1-GRP75-VDAC1 complex in AML12 cells transfected with MDM2 vector. D Confocal microscopy images showing the colocalization of ER and mitochondria. Colocalization is indicated by the merge of ER Tracker Blue-White and Mito-Tracker Green, and Pearson’s coefficient graph quantifies the colocalization (× 1200; scale bar, 5 μm; n = 4 per group). E Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1) in homogenates and MAMs fractions from AML12 cells. β-tubulin served as a loading control. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s., not significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Inhibition, Expressing, Transfection, Confocal Microscopy, Construct, Western Blot, Control, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Knockdown MDM2 and IP3R1 inhibited Ca 2+ dyshomeostasis and mitochondria dysfunction caused by PA stimulation in hepatocytes. A , B Measurements of mitochondrial Ca 2 ⁺ levels in AML12 cells transfected with MDM2 or IP3R1 vectors under BSA or PA exposure for 24 h. The upper panel shows the time course of pCAG-Mito-Gcamp5G fluorescence, recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak values are shown in the right panels ( n = 3 per group). C Western blot analysis of mitochondrial respiratory chain proteins (ATP5A, UQCRC2, NDUFB8), with β-tubulin as a loading control. D Rhodamine 123 staining and BODIPY 493/503 staining for analyzing mitochondrial membrane potential and lipid droplets. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., not significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Knockdown, Transfection, Fluorescence, Injection, Western Blot, Control, Staining, Membrane

Journal: Human Reproduction (Oxford, England)

Article Title: The role of small extracellular vesicle-miRNAs in endometriosis

doi: 10.1093/humrep/dead216

Figure Lengend Snippet: Studies investigating the differential expression of sEV-miRNA.

Article Snippet: 13 , Uterine aspirate fluid , Human samples: Endometriosis (stage III–IV, endometrioma, diagnosed via laparoscopy), n=22 Control (including simple ovarian cyst, uterine leiomyoma, tubal ligation), n=25 *Mentioned about that findings were regardless of secretory or proliferative phases in the results section but did not declare number of samples that were of proliferative or secretory. , Size exclusion chromatography exosome isolation kit (Echobiotech, Beijing, China) , NTA : Median size: 111.7 nm and the proportion of the main peak was 94.1%. Visualised via TEM . , WB : CD63, TSG101, and HSP70 positive. Calnexin negative. , Microarray analysis of miRNA from sEVs and endometrial tissues from uterine aspirate fluid, followed by qRT-PCR . sEV uptake experiments with JNK activator anisomycin by M0 macrophages followed by flow cytometry , WB, and MAPK phosphorylation antibody array . Transwell migration and invasion assay : Co-culture of endometrial tissues of control group and sEV-treated macrophages. Transfection of M0 macrophages with hsa-miR-210-3p mimic/inhibitor/corresponding NC followed by qRT-PCR and flow cytometry . , ↑ hsa-miR-210-3p Regardless of secretory or proliferative phases , Jiang et al. (2022) .

Techniques: Expressing, Isolation, Concentration Assay, Western Blot, Microarray, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Extraction, Transfection, Transmission Electron Microscopy, Sequencing, Flow Cytometry, Reporter Gene Assay, Zeta Potential Analyzer, Cell Culture, In Situ, Hybridization, Wound Healing Assay, Marker, Ligation, Migration, Size-exclusion Chromatography, Ab Array, Invasion Assay

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Normal human female karyotype prepared from WT and LRRK2 G2019S iPSCs. Cytogenetic analysis was performed on 20G-banded metaphase cells for each genotype. ( B ) Representative confocal images of non-isogenic iPSCs reprogrammed from dermal fibroblasts collected from a healthy donor and a LRRK2 G2019S patient. Immunostaining shows expression of the pluripotency markers Nanog (red) and TRA-1-81 (green), Oct4 (red) and TRA-1-60 (green), Sox 2 (red), and SSEA4 (green). Nuclei are stained with DAPI. ( C ) Heatmap showing trilineage differentiation potential of the newly generated non-isogenic iPSCs via embryoid body formation and subsequent spontaneous differentiation. Data show RT-qPCR quantification of gene expression for known markers of the ectoderm, endoderm, and mesoderm lineages, as well as self-renewal genes, and are normalized to iPSC reference standards. ( D ) Representative heatmap showing the gene expression profile of NPCs patterned towards a midbrain fate. The left side of the heatmap identifies gene markers for specific brain regions . Successfully differentiated NPCs should show higher expression levels of genes shown in the two black boxes (midbrain and floor plate) compared to other genes ( ; ). The scale is calculated based on the 1/Δ cycle threshold (Ct) values calculated from the Ct of the gene of interest compared to the Ct of the loading control β-actin. ( E ) Confocal images of immunostained NPCs show expression of NPC markers vimentin (red), SOX1 (green), and merged vimentin (red) and SOX1 (green). The lower panel shows the expression of the markers nestin (red), notch1 (green), and merged nestin (red) and notch1 (green).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Immunostaining, Expressing, Staining, Generated, Quantitative RT-PCR

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Astrocytes were prepared from induced pluripotent stem cells (iPSCs) carrying the Parkinson’s disease (PD) mutation LRRK2 G2019S or its isogenic control. Confocal images of immunostained iPSC-derived astrocytes show expression of astrocyte markers GFAP (green), vimentin (red), merged GFAP (green) and vimentin (red) with the nuclear marker DAPI (blue), and merged astrocyte marker CD44 (red) with DAPI (blue). ( B ) Heatmap representing the hierarchical clustering of significantly upregulated and downregulated genes in LRRK2 G2019S vs. WT astrocytes using a 1.4-fold threshold for upregulated genes and a 0.7-fold threshold for downregulated genes, and a false discovery rate of 0.05. Sequencing counts were normalized using the median of ratios method and calculated by the EBseq package in R, as described in Materials and methods, then transformed using log 10 (1 + normalized sequencing counts). The genes are separated into three categories based on their log 10 (1 + normalized sequencing counts) value using k-means clustering with k = 3 to reveal groups of low-, moderate-, or high-expression genes. Genes encoding exosome-related components are indicated on the right (purple lines, with each indicating one gene). ( C ) Heatmap representing the differential expression of 20 genes encoding exosome components in WT and LRRK2 G2019S astrocytes, across three independent biological replicates (labeled 1–3). The values represent the log 10 (1 + normalized sequencing counts) transformation as described in Materials and methods, log 2 FC represents LRRK2 G2019S vs. WT fold change in gene expression. Genes are sorted by their log 2 FC value in order of descending fold change. ( D–H ) Gene ontology analysis of non-isogenic and isogenic lines showing upregulated components identified by RNA sequencing, and Benjamini–Hochberg adjusted p-values were obtained from the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool ( D, F ). Gene expression validation by qPCR of two exosome components, Rab27b ( E, G ) and CD82 ( H ) in non-isogenic and isogenic lines. Data are from three ( B, C, D, F ) four ( G, H ), or five ( E ) independent biological replicates; error bars represent mean + standard error of the mean (SEM). Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal standard deviation (s.d.) (*p≤0.05, **p<0.01).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Mutagenesis, Derivative Assay, Expressing, Marker, Sequencing, Transformation Assay, Labeling, RNA Sequencing Assay, Two Tailed Test, Standard Deviation

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Histogram representing the proportion of astrocytes expressing the marker CD44 by flow cytometry in cultures prepared from human fetal midbrain astrocytes (89.55% CD44 + cells) or iPSC-derived astrocytes (88.54% CD44 + cells). A total of 10,000 events were recorded for each experimental condition (Ai). CD44 gene expression in iPSCs, neural progenitor cells (NPCs), iPSC-derived astrocytes (iAstrocytes), or human fetal midbrain astrocytes (hMidbrain) collected by RNA-seq and expressed as CPM values (Aii). ( B ) Quantification of GFAP + astrocytes in cultures differentiated from WT or LRRK2 G2019S iPSCs. Data are from two independent biological replicates, and at least 260 cells were counted per experimental condition (Bi). Gene expression of astrocyte markers in astrocytes derived from isogenic and non-isogenic iPSC lines, as well as human fetal midbrain astrocytes to use as a reference. Data were collected by RNA-seq and shown as the mean log 2 (CPM) values (Bii). ( C, D ) To confirm the successful differentiation of iPSCs into specific cell types, we performed RNA-seq of iPSCs, NPCs, iPSC-derived astrocytes, and human fetal astrocytes. RNA-seq data was analyzed by principal component analysis (PCA) ( E ) and a heatmap representing the unsupervised cluster analysis was generated ( F ); data are from two (iPSCs) or three (NPCs, iPSC-derived astrocytes and fetal astrocytes) independent biological replicates. ( E, F ) Representative wide-field images showing transient calcium signals using the green Fluo-4 indicator ( E ) and internalization of green-labeled synaptosomes ( H ) in WT and LRRK2 G2019S iPSC-derived astrocytes. Arrowheads in ( E ) show cells with transient increase in Fluo-4 signal indicative of calcium influx. Abbreviation: CPM, counts per million.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Expressing, Marker, Flow Cytometry, Derivative Assay, RNA Sequencing Assay, Generated, Labeling

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Transmission electron microscopy (TEM) images of MVBs in WT and LRRK2 G2019S astrocytes. For illustration purposes, the red boxes in the top panel indicate MVBs in the cytoplasm of astrocytes and the lower panel shows a zoomed-in view of the MVBs. ( B, C ) Quantification of mean area ( B ) and size distribution ( C ) of MVBs identified in TEM images of WT and LRRK2 G2019S astrocytes. Data are sampled from at least 20 cells (≥40 MVBs) in each experimental condition; error bars represent mean + SEM for two independent biological samples ( B ). Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (****p<0.0001). ( D ) Representative confocal images of astrocytes labeled by immunofluorescence with the exosome marker CD63 (green) and the astrocyte marker CD44 (red). ( E ) Electron microscopy image showing immunogold labeling of CD63 (large gold) in astrocytes. Dashed lines delineate MVB membranes.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transmission Assay, Electron Microscopy, Two Tailed Test, Labeling, Immunofluorescence, Marker

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A, B ) Quantification of CD63 gold particles shown as the number of particles per µm 2 multivesicular body (MVB) ( A ) and relative frequency ( B ) in WT vs. LRRK2 G2019S astrocytes. Error bars represent mean + SEM. ( C ) Transmission electron microscopy (TEM) image of an astrocyte and astrocyte-secreted EVs. The dashed line indicates the astrocyte cell membrane, and the arrows indicate EVs. The red box and the zoomed-in view show EVs that appear to bud from the astrocyte membrane (white arrowheads). ( D ) Identification of exosome markers in EV-enriched fractions obtained from astrocyte conditioned media (ACM) using an exosome antibody array. Each circle on the membrane represents a preprinted antibody spot marker of exosome or cellular contaminant, and the table details the name of each antibody marker spotted on the membrane. ( E ) Cryo-electron microscopy (cryo-EM) images of astrocyte-derived EVs that display an unusual morphology. ( F ) Quantification of the number of CD63 + EVs secreted in WT and LRRK2 G2019S ACM by ELISA using ELISA standards calibrated by nanoparticle tracking analysis (NTA) as discussed in Materials and methods. Data are from at least three independent biological replicates; error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transmission Assay, Electron Microscopy, Ab Array, Marker, Cryo-EM Sample Prep, Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Transmission electron microscopy (TEM) images demonstrate that induced pluripotent stem cell (iPSC)-derived astrocytes actively produce and secrete EVs by exocytosis. The dashed line delineates the plasma membrane. The white arrows indicate EVs. ( B ) Overview of the procedure to isolate EVs by ultracentrifugation. ACM: astrocyte conditioned medium; Cryo-EM: cryogenic electron microscopy. ( C ) Nanoparticle tracking analysis (NTA) quantification of the number of particles (i.e., EVs) isolated in WT or LRRK2 G2019S ACM by ultracentrifugation as described in ( B ). ( D ) Graph showing the distribution of isolated EVs by particle size. Data in ( C, D ) are from two independent biological replicates; error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant). ( E–G ) Secreted EVs were imaged by cryo-EM ( E ) and their diameter ( F ) and morphology ( G ) was analyzed. Data are from ≥177 EVs isolated from ACM taken from 30.4 × 10 6 plated astrocytes for each experimental condition; error bars are mean + SEM for three independent biological replicates. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (*p≤0.05).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transmission Assay, Electron Microscopy, Derivative Assay, Cryo-EM Sample Prep, Isolation, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Representative images of WT and LRRK2 G2019S isogenic astrocytes labeled by immunofluorescence with CD63 (green), LRRK2 (purple), and DAPI nuclear stain (blue) ( Ai, Aiv ). The images were analyzed using Imaris software to identify CD63 + MVBs (green surfaces) colocalized with LRRK2 (purple dots) and show the localization of the nucleus (DAPI, blue) ( Aii, Av ). Zoomed-in images shows two populations of CD63 + surfaces: CD63 + /LRRK2 + (red arrowhead), and CD63 + /LRRK2 - (white arrowhead) ( Aiii, Avi ). ( B ) Percentage of CD63-labeled surfaces that are also LRKK2 positive in WT and LRRK2 G2019S astrocytes, quantified with Imaris software using object-based colocalization. Data are from three independent biological replicates, and ≥40 astrocytes (>3000 MVBs) were analyzed per experimental condition; error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant). ( C ) Immunogold electron microscopy shows the presence of LRRK2 (small gold, red arrowheads) inside and in the vicinity of CD63 + MVBs (large gold) in WT and LRRK2 G2019S astrocytes. The dashed lines indicate the contour of MVBs. ( D ) Distribution of CD63 + MVBs according to their number of internal LRRK2 gold particles. Data are sampled from at least 20 astrocytes (≥59 MVBs) in each experimental condition. The distribution is significantly different in LRRK2 G2019S astrocytes compared to WT astrocytes (p-value = 0.0084, chi-square test). ( E ) Quantification of the amount of LRRK2 in WT or LRRK2 G2019S EV-enriched fractions by ELISA. Data are from at least three independent biological replicates; error bars represent mean + SEM; statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Labeling, Immunofluorescence, Staining, Software, Two Tailed Test, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of WT and LRRK2 G2019S astrocytes labeled with the exosome marker CD63 (green), the astrocyte marker CD44 (red), and the nuclear marker DAPI (dark blue). The bottom images show the corresponding Imaris software rendering of CD63 + MVBs, color-coded by distance to the nucleus, from blue (closest) to white (farthest). The plain white lines indicate the cell boundary (outer line) and nucleus (inner circle). ( B–E ) Quantification of the distance of CD63 + MVBs from the nuclear membrane in WT and LRRK2 G2019S isogenic ( B, C ) or non-isogenic ( D, E ) astrocytes using Imaris software ‘vesicles distance to closest nucleus’ calculation. The violin plot shows the median (blue dashed line) and interquartile range (red solid line) ( B, D ). Data are from three independent biological replicates, 40–70 astrocytes (>3300 MVBs) were analyzed for each experimental condition. Statistical analysis was performed using a Mann–Whitney test (****p<0.0001).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Immunofluorescence, Labeling, Marker, Software, MANN-WHITNEY

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Immunogold labeling of phospho-S129 alpha-synuclein (p-αSyn, small gold, red arrowheads) and CD63 (large gold) shows localization of p-αSyn inside and in the vicinity of MVBs in astrocytes. The low abundance of small gold particles in the control sample is consistent with the observation that healthy brain tissues contain low levels of p-αSyn . The dashed lines indicate the boundary of the MVBs. ( B ) Distribution of CD63 + MVBs according to their number of internal p-αSyn gold particles, in WT and LRRK2 G2019S astrocytes. Data are sampled from at least 20 astrocytes (30–79 MVBs) for each experimental condition. The distribution is significantly different in LRRK2 G2019S astrocytes compared to WT astrocytes (p-value=0.0014, chi-square test). ( C ) Quantification of the amount of αSyn in WT and LRRK2 G2019S extracellular vesicle (EV)-enriched fractions by ELISA. Data are from seven independent biological replicates; error bars represent mean + SEM; statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) WT dopaminergic neurons were transduced with rh10-CAG-tdTomato (red), and WT or LRRK2 G2019S astrocytes were transduced with lenti-CD63-GFP (green) to produce green-labeled exosomes. Neurons and astrocytes were co-cultured, and uptake of CD63-GFP exosomes by neurons was monitored by live-cell confocal microscopy, followed by deconvolution and Imaris modeling. ( B ) Confocal images of tdTomato neurons ( Bi ), CD63-GFP astrocytes ( Bii ), and the merged image ( Biii ). The corresponding Imaris software rendering represents tdTomato neurons in yellow ( Biv ), and the CD63-GFP exosomes in blue (outside the neurons) or purple (inside the neurons) ( Bv ). A transverse view of a neuron shows purple-labeled exosomes inside the somas and neurites ( Bvi ). ( C ) Quantification of the percentage of neurons with internalized WT or LRRK2 G2019S CD63-GFP exosomes at the time of live-cell imaging. ( D ) Quantification of the number of CD63-GFP exosomes per unit of neuronal volume. ( E ) Confocal images of tdTomato neurons co-cultured with WT or G2019S CD63-GFP isogenic astrocytes, and the corresponding Imaris software rendering representing neurons in white and CD63-GFP exosomes in red. White arrowheads show exosomes inside neurites. Scale bar: 5 µm. ( F ) Quantification of the number of CD63-GFP exosomes per unit of soma or neurite volume. For all datasets: data are from three independent biological replicates, ≥80 neurons were analyzed for each experimental condition. The scatter plot shows the median value. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. ( C, D ), or one-way ANOVA with Newman–Keuls multiple comparisons ( E ) (ns: not significant, *p≤0.05).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transduction, Labeling, Cell Culture, Confocal Microscopy, Software, Live Cell Imaging, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Confocal images showing neurons labeled by immunofluorescence with the pan-neuronal and dendrite marker MAP2 (red), the marker for dopaminergic neurons TH (green), and the merged images (MAP2, red, and TH, green). MAP2: microtubule-associated protein 2; TH: tyrosine hydroxylase. ( B–F ) Quantification of neuron viability (number of dopaminergic neurons remaining in culture) ( B ), average dendrite length ( C, E ), and dendrite length distribution ( D, F ) after 14 days in culture with WT or LRRK2 G2019S isogenic ( B–D ) or non-isogenic ( E, F ) astrocytes. Viability data are from five ( B, D ) independent biological replicates, and at least 500 neurons were counted per experimental condition and biological replicate. WT and 530 LRRK2 G2019S neurons were counted ( B ). Dendrite length data are from three independent biological replicates, and more than 300 ( E, F ) or 500 ( C, D ) neurites were measured for each experimental condition. ( G ) Confocal images showing WT mouse primary neurons co-cultured with WT or LRRK2 G2019S mouse primary astrocytes. Neurons are labeled by immunofluorescence and images show MAP2 (red), TH (green), and the merged images (MAP2, red, and TH, green). White arrowheads mark TH + /MAP2 + dopaminergic neurons. ( H, I ) The cells were scored for TH + /MAP2 + dopaminergic ( H ) or TH - /MAP2 + non-dopaminergic neuron survival ( I ) after 14 days co-culture with WT or LRRK2 G2019S astrocytes. ( J, K ) LRRK2 G2019S mouse primary neurons were co-cultured with mouse WT or LRRK2 G2019S astrocytes as described in ( H, I ) and scored for relative viability of TH + /MAP2 + dopaminergic ( J ) or TH - /MAP2 + non-dopaminergic neurons ( K ). Viability data for all mouse primary culture experiments are from three independent biological replicates, and at least 200 neurons were counted for each experimental condition and biological replicate. For all datasets, error bars represent mean + SEM; statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (*p≤0.05, **p<0.01, ****p<0.0001). Results shown in panel ( B ) support similar observations recently documented in a non-isogenic induced pluripotent stem cell (iPSC)-based model system .

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Labeling, Immunofluorescence, Marker, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A–D ) WT dopaminergic neurons were cultured for 14 days with basal medium (-), WT or LRRK2 G2019S astrocytes conditioned media (ACM), and the resulting dendrite length average ( A, C ) and distribution ( B, D ) were quantified. Data in ( A, B ) were collected using the isogenic induced pluripotent stem cell (iPSC)-based model, data in ( C, D ) used the primary mouse culture system. The data are from three independent biological replicates, and ≥450 ( A, B ) or 170 ( C, D ) neurites were measured per experimental condition. ( E, F ) Quantification of the average ( E ) and distribution ( F ) of dendrite lengths of WT dopaminergic neurons cultured for 14 days with basal medium (-), or WT or LRRK2 G2019S extracellular vesicle (EV)-free ACM. Data are from four independent biological replicates, and ≥650 neurites were measured for each experimental condition. ( G, H ) Quantification of the average ( G ) and distribution ( H ) of dendrite lengths of WT dopaminergic neurons cultured for 14 days with basal medium (-), or WT or LRRK2 G2019S EV-enriched fractions. Data are from three independent biological replicates, and ≥450 dendrite were measured for each experimental condition. For all datasets, error bars represent mean + SEM, and statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. ( C ) or one-way ANOVA with Tukey’s multiple comparisons correction ( A, E, G ) (ns: not significant, *p≤0.05, ****p<0.0001).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Cell Culture, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: Gene ontology analysis of isogenic ( Ai ) and non-isogenic ( Aii ) LRRK2 G2019S vs. WT induced pluripotent stem cell (iPSC)-derived astrocytes showing downregulated components identified by RNA sequencing. Benjamini–Hochberg adjusted p-values were obtained from the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. ( B, C ) Heatmaps showing top significantly downregulated genes associated with lipid biogenesis ( B ) and cell survival ( C ) in LRRK2 G2019S vs. WT astrocytes using a 0.7-fold threshold and a false discovery rate of 0.05. Data shows log 2 (CPM) values calculated for WT and LRRK2 G2019S astrocytes prepared using the isogenic or non-isogenic lines, as well as human midbrain fetal astrocytes to use as a reference. Abbreviation: CPM, counts per million.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Derivative Assay, RNA Sequencing Assay

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet:

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Magnetic Beads, Knock-In, Labeling, Purification, SNP Genotyping Assay, Recombinant, Sequencing, Software

Journal: bioRxiv

Article Title: A single-cell fixed RNA profiling of liver fibrosis progression and regression reveals SEMA4D and LMCD1 as key mediators of fibrogenesis

doi: 10.1101/2025.06.12.657772

Figure Lengend Snippet: a UMAP showing 9 subpopulations of LSECs from control, cirrhosis, and the regression group, colored by cluster. b UMAP showing LSECs from control, cirrhosis, and regression group, colored by group. c Barplot showing group composition breakdown per cluster (fraction of total cell count per cluster) d Stacked violin plot showing markers of LSECs landscape and mixed clusters e Dot plot represented pathways distinguishing each LSECs subpopulation by ClusterProFiler. Dot sizes corresponded to the ratio of pathway genes found in the dataset. Colors corresponded to p-value adjustment. Genes sets used for the analysis were taken from the Gene Ontology (GO) dataset. f Heatmap displayed up-regulation of ECM-degrading enzymes (Ctsl, Ctsd, Ctsf, Ctsz, and Mmp14), interferon-stimulated genes (Ifitm1–3), and antioxidant genes (Selenop, Tcn2, and Txnip) in the regression group. g Heatmap of the top genes that were differentially expressed along the pseudotime, colored by gene expression. h Flow cytometry showing CD146 and CD45 positive cells among control, cirrhosis and the regression group (upper panel). Scanning electron microscopy (SEM) images showed morphology and fenestration of LSECs in liver tissues from control, cirrhosis and regression. Scale bar 2.5 𝜇𝜇m. SEM image showed morphology and fenestration of isolated LSECs from control, cirrhosis and the regression group after culturing 6 hours in medium. Scale bar 2.5 𝜇𝜇m. Double IF staining for P21 (red), CD31 (green), and DAPI (blue) for nuclear staining confirms colocalized of P21-CD31 in cirrhosis livers (white arrow). IHC staining showing expression of Lymphatic vessel endothelial receptor 1 (LYVE1) in healthy, fibrosis and regressed group (lower panel). Scale bar 50 𝜇𝜇m. i Cytokine array membrane of soluble factors in cultured medium of LSECs isolated from control, cirrhosis and the regression group (left panel) and heatmap showing the quantification of mean pixel density (MPI, right panel). The soluble factors with predominant change in TAA were marked with red boxes, the soluble factors with predominant change in healthy control (CONT) were marked with blue boxes. j Bubble colors and sizes represent the per-sample scaled product of normalized ligand and receptor pseudo-bulk expression and sufficient presence L-R. Heatmap colors indicated the ligand activity and scaled ligand activity in the receiver.

Article Snippet: Secreted proteins in culture medium from isolated LSECs, macrophages, cholangiocytes and mouse serum were screened by using Proteome Mouse XL Cytokine Array Kit (#ARY028, R&D system, Minneapolis, MN, USA), which can detect 111 soluble proteins.

Techniques: Control, Cell Counting, Gene Expression, Flow Cytometry, Electron Microscopy, Isolation, Staining, Immunohistochemistry, Expressing, Membrane, Cell Culture, Activity Assay

Journal: bioRxiv

Article Title: A single-cell fixed RNA profiling of liver fibrosis progression and regression reveals SEMA4D and LMCD1 as key mediators of fibrogenesis

doi: 10.1101/2025.06.12.657772

Figure Lengend Snippet: a UMAP showing 6 subpopulations of CHOLs, colored by cluster. b UMAP showing CHOLs from control, cirrhosis, and regression group, colored by group. c Barplot shows group composition breakdown per cluster (fraction of total cell count per cluster), colored by group. d Stacked violin plot showing markers of cholangiocytes and mixed clusters. e Multi-ridge plots show the comparison of normalized enrichment score (NES) of selected gene set by scGSVA, colored by CHOLs subpopulation. f, g Pseudotime trajectory mapped to cholangiocytes UMAP coordinates. Direction of arrows indicates predicted cell state progression from fibrosis to regression phase (f) and heatmap (g) showing the dynamics of differentially expressed genes between two cell states. Genes (rows) and cells (columns) are ordered according to the pseudo-time trajectory. h Volcano plot showing differential gene expression of cholangiocytes in control and fibrosis group (upper panel), regressed and fibrosis group (lower panel). Gene upregulated or downregulated by more than 2-fold and significant to p < 0.05 is shown in red. i Quantification of Mmp7 expression by qRT-PCR in CHOLs isolated from control, fibrosis and regression group (left panel). Gapdh was used as internal control. Quantification of Mmp7 concentration in serum of control, cirrhosis, and regression group (right panel). Data represent the means ± SD, p- values were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001. j Dot plot showing inflammatory-related genes of CHOLs cells subpopulation, colored by gene expression. The size of the dot corresponds to the percentage of cells expressing the gene in each CHOLs subpopulation. k Cytokine array of soluble factors in cultured media from cholangiocytes isolated from control, cirrhosis, and in the regression group, the soluble factors with predominant change were marked with red boxes or circles (left panel). Heatmap showing the soluble factors expression of each group by calculating mean pixel density (MPI), colored by MPI (right panel).

Article Snippet: Secreted proteins in culture medium from isolated LSECs, macrophages, cholangiocytes and mouse serum were screened by using Proteome Mouse XL Cytokine Array Kit (#ARY028, R&D system, Minneapolis, MN, USA), which can detect 111 soluble proteins.

Techniques: Control, Cell Counting, Comparison, Gene Expression, Expressing, Quantitative RT-PCR, Isolation, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: A single-cell fixed RNA profiling of liver fibrosis progression and regression reveals SEMA4D and LMCD1 as key mediators of fibrogenesis

doi: 10.1101/2025.06.12.657772

Figure Lengend Snippet: a UMAP showing NK&T cells from control, cirrhosis, and regression group, colored by group. b UMAP showing 9 subpopulations of NK&T cells, colored by cluster. c Barplot shows group composition breakdown per cluster (fraction of total cell count per cluster), colored by group. d Stacked violin plot showing expression markers of NK&T cells. e Barplot showing top 4 GO gene set enrichment analysis of NK&T subclusters S5, S6; compared by -log10(PVAL) of each pathway. f Dot plot showing cytokine-related genes of NK&T cells subpopulation, colored by gene expression. The size of the dot corresponds to the percentage of cells expressing the gene in each cluster. g Transcription factor analysis by Dorothea, colored by average expression (upper panel); Transcription factor activity of Mef2a, Mef2b, Mef2c in NT&T cells subpopulation, colored by gene expression. h Interaction showing CCL (upper panel) and CXCL (lower panel) signaling pathways network interaction among all cell types in fibrosis and regressed livers i Gating strategy for NK&T cell subpopulations in control, cirrhosis, and in the regression group with CD45+CD4+CD8+FOXP3+NK1.1+KLRG1+.

Article Snippet: Secreted proteins in culture medium from isolated LSECs, macrophages, cholangiocytes and mouse serum were screened by using Proteome Mouse XL Cytokine Array Kit (#ARY028, R&D system, Minneapolis, MN, USA), which can detect 111 soluble proteins.

Techniques: Control, Cell Counting, Expressing, Gene Expression, Activity Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: A . Simplistic schematic of ribosome profiling technique. B . Scatterplot of differential expression between SW480 cells with and without IMP1 deletion. The log2 fold change between ribosome-bound RNAs (ribosome protected fragments, or RPF) and total mRNA is plotted. The plot indicates that IMP1 regulates both mRNA abundance and translation. C . Scatterplot of genes with significant (in blue) differential translational efficiencies between SW480 cells with and without IMP1 deletion. Translation efficiencies of transcripts are calculated as the ratio of reads of ribosome-protected fragments to the reads in total mRNA abundance. D . Pathway analysis using Toppgene gene enrichment analysis software of differentially expressed genes from C to define which signaling/effector pathways are enriched with IMP1 deletion.

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Quantitative Proteomics, Software

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: A. Paneth cells from Imp1 WT and Imp1 ΔIEC mice were evaluated histologically using IF for lysozyme (LYZ). E-cadherin (ECAD) staining was used to demarcate individual epithelial cells. Note presence of diffuse lysozyme staining in Imp1 ΔIEC mice. B . Published lysozyme scoring was utilized to evaluate specific Paneth cell phenotypes. Imp1 ΔIEC mice exhibit a significant shift from normal to diffuse lysozyme phenotype (n= 4 mice per genotype). C . Transmission electron microscopy revealed an abundance of small, electron dense granules in Imp1 ΔIEC mice. D . To confirm a direct role for IMP1 knockdown to induce autophagy, we evaluated epithelial cells from colon in Imp1 WT and Imp1 ΔIEC mice. This confirmed a robust increase in LC3 and decrease in p62, which together indicate enhanced flux. Blots are representative of 3 independent experiments. E . Live cell staining of autophagic structures with the cationic amphiphilic tracer dye CytoID indicated a significant increase in autophagic vesicles in crypts from Imp1 ΔIEC mice (n=8) compared to Imp1 WT mice (n= 7) using flow cytometry. There was a modest increase in basal CytoID in Imp1 ΔIEC enteroids compared to controls, as seen with FACS analysis of isolated crypts from Imp1 ΔIEC mice, and both genotypes exhibited an increase in CytoID puncta following 12Gy radiation treatment. (All data are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; by ordinary one-way ANOVA test or standard t-test).

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Staining, Transmission Assay, Electron Microscopy, Knockdown, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: We utilized computational predictions, analyses of published CLIP-Seq data and ribonucleoproteinimmunoprecipitation (RIP) to identify direct interactions between IMP1 and autophagy transcripts. A . Caco2 cells transfected with IMP1 siRNA demonstrated a robust increase in LC3-I/LC3-II indicating enhanced flux. Blots are representative of 3 independent experiments. B . catRAPID Transcript Score. For each predicted transcript, we measured the IMP1 interaction propensity with respect to the negative control IgG. Negative targets (ITGA7 and TNFRSF1B) were also evaluated. C . We retrieved CLIP scores from published eCLIP data against the same set of autophagy-related transcripts analyzed in B and found a strong correlation between catRAPID scores and eCLIP data (r =0.9838465 Pearson correlation coefficient). The CLIP data scores were calculated as total number of reads corresponding to the transcript divided by the length of the different isoforms. D . We evaluated binding of endogenous IMP1 to autophagy transcripts using RIP assays in Caco2 cells. Specific enrichment of IMP1 was confirmed by IP with either IMP1 or control IgG antibodies followed by western blot for IMP1. Enrichment of target transcripts over control is represented relative to negative target, TNFRSF1B. Positive controls were PTGS2 and ACTB. vs. negative target by 1-way ANOVA. n=3 independent experiments. E . Representative western blot showing upregulation of Atg3 and Atg5 in colon epithelium of Imp1 ΔIEC mice as compared to controls. (All data are expressed as mean ± SEM. *, p < 0.05; by ordinary oneway ANOVA test).

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Transfection, Negative Control, Binding Assay, Control, Western Blot

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: A . Simple schematic of the experiment. Mice are treated with 12Gy whole body radiation and all the analyses are performed at day 4 following radiation. B . Imp1 deletion confers protective effects following irradiation, which is reversed in the context of Atg7 deletion. Imp1 ΔIEC mice lost significantly less weight at sacrifice following irradiation than controls (18.83±0.98% in Imp1 ΔIEC mice(n=12) versus 23.34±0.56% mean weight loss in controls (n=14). This phenotype was abrogated in Imp1 ΔIEC Atg7 ΔIEC mice (23.5±1.05% mean weight loss, n=5). For untreated animals, there was no significant difference in mean body weights between groups (not shown). C&D . Analysis of EdU+, S-phase cells revealed similar staining in all non-IR mice (not shown); however, there was a robust increase in EdU+ regenerative crypt foci at 4 days following irradiation in Imp1 ΔIEC mice compared to Imp1 WT mice, and this effect was abolished in Imp1 ΔIEC Atg7 ΔIEC mice. (n=4 mice per genotype, 20–30 HPF per animal). (All data are expressed as mean ± SEM. *, p < 0.05; by ordinary one-way ANOVA test or standard t-test).

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Irradiation, Staining

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: Mice were given 5% DSS in drinking water for 5 days followed by 4 days of recovery. B . Mice with Imp1 deletion lost significantly less weight as compared to controls. Imp1 ΔIEC Atg7 ΔIEC mice lost weight and became moribund more rapidly than Imp1 WT and Imp1 ΔIEC mice, requiring sacrifice prior to the recovery period. C . Imp1 ΔIEC mice show significantly less total colitis (11.14 ± 1.933, n=7; As scored blinded by a pathologist) as compared to Imp1 WT mice (17.75 ± 2.144, n=8). D . Imp1 ΔIEC mice show significantly less epithelial loss (5.286 ± 0.865, n=7) as compared to Imp1 WT mice (9.375 ± 1.375, n=8). E . Mice were given 3 cycles of 2.5% DSS in drinking water for 5 days followed by a week of recovery. Mice were analyzed at day 36. F . Imp1 ΔIEC mice show significantly less total colitis (7.769 ± 0.4824, n=13; As scored blinded by a pathologist) as compared to Imp1 WT mice (9.714 ± 0.8371, n=7). G . Imp1 ΔIEC mice show significantly less hyperplasia (1.615 ± 0.1404, n=13) as compared to Imp1 WT mice (2.286 ± 0.2857, n=7). H . Imp1 ΔIEC mice show significantly less inflammation score (6.231 ± 0.3608, n=13) as compared to Imp1 WT mice (8 ± 0.6901, n=7). I . Imp1 ΔIEC mice show significantly less mono-nuclear cell infiltration (1.077 ± 0.076, n=13;) as compared to Imp1 WT mice (1.714 ± 0.2857, n=7). J . qPCR data showing expression of different cytokines in colon epithelium of Imp1 WT and Imp1 ΔIEC mice at day 36 after chronic DSS treatment. (All data are expressed as mean ± SEM. *, p < 0.05; by standard t-test).

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Expressing

Journal: bioRxiv

Article Title: Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

doi: 10.1101/368050

Figure Lengend Snippet: A .qPCR analysis for IMP1 expression in colon biopsy samples from adult Crohn’s disease (CD) patients. IMP1 expression is >5 fold higher in CD samples (5.314 ± 1.807, n=8) as compared to control samples (1 ± 0.1245, n=7). B . Representative immunohistochemistry demonstrating IMP1 expression in colon biopsy samples from CD patients and normal adults. IMP1 expression is higher in CD samples (Scale bars = 500m). C . Differential gene expression analysis of pediatric CD patient colon samples (n=180) show increased (>4 fold) IMP1 expression as compared to non-inflammatory bowel disease (IBD) (n=43) pediatric samples. (All data are expressed as mean ± SEM. *, p < 0.05; by standard t-test).

Article Snippet: IMP1 was deleted in SW480 cells by cotransfecting cells with IMP1 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz; sc-401703) and IMP-1 HDR Plasmid (h) (Santa Cruz; sc-401703 HDR) followed by sorting and clonal expansion of RFP+ve cells.

Techniques: Expressing, Control, Immunohistochemistry, Gene Expression

Journal: iScience

Article Title: Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy

doi: 10.1016/j.isci.2024.108807

Figure Lengend Snippet: Effect of tumor-derived EVs on bone-marrow-derived macrophage cytokine secretion, phagocytosis, and PD-L1 expression (A) Bone-marrow-derived macrophages (BMDMs) were exposed to palm.tdTomato (pTom)-labeled EVs, fixed and stained for F4/80 (macrophage marker; green) and DAPI (blue), and EV uptake was analyzed by fluorescence analysis. (B‒D) BMDM were exposed to EVs derived from untreated (BMDM+EV) or irradiated (BMDM+irEV) CT-2A cells. Schematic of experimental setup (B). GSEA pathway analysis showing significantly upregulated immune-related pathways (n = 2) in CT-2A EV and irEV-treated BMDM (C). Graph bar showing cytokine array results of BMDM exposed to 5 Gy radiation (BMDM+5 Gy), EV, or irEV depicting the top 10 differentially expressed cytokines (D). (E) BMDM exposed to EVs or irEVs were co-cultured with CT2A or 005 glioma cells labeled with pHrodo Red, pre-treated with and without anti-CD47, and analyzed for phagocytic ability by flow cytometry. Experimental setup schematic (Left). Graph depicting the phagocytosis index of BMDM under different conditions (Right). (F) BMDM exposed to 5 Gy RT or EVs/irEVs from CT-2A or 005 glioma cells were analyzed for PD-L1 levels. Gating strategy to identify BMDM (CD45 POS CD11b POS F4/80 POS ) (Left). Analysis of PD-L1 levels (gMFI) in BMDM under different conditions by flow cytometry (Right). Significance indicated as: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; one-way ANOVA. (n ≥ 3), Data presented as mean ± SEM. See also Figure S4 .

Article Snippet: The cytokine array was performed using the Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028s) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Expressing, Labeling, Staining, Marker, Fluorescence, Irradiation, Cell Culture, Flow Cytometry

Journal: iScience

Article Title: Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy

doi: 10.1016/j.isci.2024.108807

Figure Lengend Snippet:

Article Snippet: The cytokine array was performed using the Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028s) according to the manufacturer’s protocol.

Techniques: Recombinant, Modification, Saline, Electron Microscopy, Plasmid Preparation, Red Blood Cell Lysis, Live Cell Imaging, Protease Inhibitor, Staining, Sequencing, Software

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) Heat map of Affymetrix array data showing expression levels (log2 fold) of either PNUTS pre-RNA or PNUTS mRNA in control (CTRL) or hnRNP E1 knockdown (E1KD) NMuMG cells. The data were generated from triplicates samples. * Two distinct probes were used to target the spliced PNUTS RNA. (b) NCBI database accession numbers of PNUTS mRNA and PNUTS predicted lncRNA isoform in human. (c) Validation by RT-PCR analysis with primers specific to PNUTS isoforms of alternative PNUTS gene processing upon hnRNP E1 knockdown in human A549 cell line. (d) (Left) PNUTS isoform expression levels analyzed by RT-PCR in human breast tumor samples (T) or non-tumor counterparts (NT). (Right) Quantitative RT-PCR analysis of lncRNA-PNUTS, ZEB-1 and ZEB-2 expression in 24 human breast tumor samples. Relative expression levels of transcripts were calculated using the ΔCt method normalizing to GAPDH. Correlations between transcript expression levels were evaluated using Pearson correlation coefficient test. (Linear regression, df=24-2, a Pearson score > 0.515 and p<0.05 was considered as significant). Source data are available in . (e) PNUTS isoform expression screening by RT-PCR analysis in MCF10a mammary gland epithelial cells and MDA-MB-468 breast cancer epithelial cells, or in the metastasis progression model of MDA-MB-231 mesenchymal cell line (MDA-231, BOM-1833, LM2-4175). E-Cadherin (CDH1) was used as epithelial marker while vimentin (VIM) and ZEB1 were used as mesenchymal cell specific markers. (f) Map of PNUTS isoforms acquired by sequence alignment and drawn by using fancyGene online software. (g) Schematic representation of the alternative splicing region of the PNUTS variants (ASS: Alternative Splicing Site). (h) RT-PCR amplification of exon11–exon12 junction encompassing the predicted alternative splicing site using intron-flanking PCR primers as indicated in (g). (i) Northern-blot analysis of both PNUTS mRNA and lncRNA isoforms expression in control (SCR) or hnRNP E1 knockdown (E1KD) A549 cell clones.

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Expressing, Control, Knockdown, Generated, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Sequencing, Software, Alternative Splicing, Amplification, Northern Blot, Clone Assay

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) Secondary structure of the human PNUTS alternative splicing site as predicted by the Mfold algorithm (ΔG= −3.90 kcal.mol −1 ). The underlined nucleotides colored in red represent the mutant probe used for the REMSA experiment in (b) and (c). The red asterisk represents the exact alternative splicing site leading to the lncRNA-PNUTS isoform generation. (ASS: Alternative Splicing Site) (b) (Left) RNA electromobility shift assay (REMSA) experiment using either wild-type [PNUTS-BAT] or mutated [PNUTS-MUT] α-32P-labelled PNUTS alternative splicing site probes combined with control (SCR) or hnRNP E1 knockdown (E1KD) A549 cell lysates. The [PNUTS-MUT] probe was mutated by a nucleotide substitution to destroy its secondary structure. [Non specific] and [DAB2-BAT] α- 32 P-labelled probes were used as negative and positive controls respectively. [DAB2-BAT] corresponds to the BAT-sequence located on the Dab2–3'UTR already described to bind to hnRNP E1. (Right) REMSA using a combination of [PNUTS-BAT] or mutated [PNUTS-MUT] α- 32 P-labelled probes with increasing concentration of recombinant hnRNP E1 protein purified from e. coli bacteria. (c) Time course experiment using RT-PCR analysis of PNUTS gene processing after addition of 5ng.mL −1 of TGFβ. (d) Confocal microscopy imaging of the hnRNP E1 nucleocytoplasmic shuttling by addition of 5µg.mL −1 of Actinomycin D for 3h in A549 and NMuMG cells cultures. Scale bar: 10µM (e) Characterization of the nucleocytoplasmic transportation of hnRNP E1 following Act.D treatment by using cell fractionation and subsequent western-blot analysis of hnRNP E1 expression. To check the fractions purity, GAPDH and PARP were used as cytoplasmic and nuclear compartment markers respectively. (f) Time course experiment using RT-PCR analysis of PNUTS predicted-lncRNA alternative splicing activation upon addition of 5µg.mL −1 of Act.D in control (CTRL) or hnRNP E1 silenced (E1KD) A549 and NMuMG cells. (g) Inhibition of alternative splicing induced by Act.D in A549 cells using and antisense oligonucleotide (ASO) targeting the alternative splicing site of PNUTS. GAPDH was used as a loading control. Unprocessed original scans of blots are shown in

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Alternative Splicing, Mutagenesis, Electro Mobility Shift Assay, Control, Knockdown, Sequencing, Concentration Assay, Recombinant, Purification, Bacteria, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy, Imaging, Cell Fractionation, Western Blot, Expressing, Activation Assay, Inhibition

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Fractionation, Reverse Transcription Polymerase Chain Reaction, Expressing, Reverse Transcription, Confocal Microscopy, Imaging, Cotransfection, Construct, Activation Assay, Quantitative RT-PCR, In Silico, Binding Assay, Incubation, In Vitro, Labeling, Extraction, Transfection, Concentration Assay, Over Expression, Control

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) MDA-231-LM2-4175 cells stably silenced for lncRNA-PNUTS were analyzed by immunofluorescence (left) using antibodies against vimentin (green), E-cadherin (red) and merged with DAPI (blue). Scale bar: 50µM. lncRNA-PNUTS silencing was monitored by RT-PCR (right, top). Invasive capacities of control (SCR-shRNA) or lncRNA-PNUTS silenced (PNUTS shRNA) cells were monitored in modified Boyden chamber assay (right, bottom). (Mean ± s.d., n= 3 independent experiments per condition). Source data are available in . (b) A549 and NMuMG cells stably overexpressing lncRNA-PNUTS were analyzed using bright-field microscopy. hnRNP E1 knockdown (E1KD) cells were used as controls. Scale bar: 100µM. (c) Western-blot (top) and RT-PCR (bottom) analysis of E-cadherin, vimentin and lncRNA-PNUTS in A549 and NMuMG cells overexpressing lncRNA-PNUTS. (d) RT-PCR analysis of several EMT-related transcription factors in A549 cells stably overexpressing lncRNA-PNUTS. (e) Schematic outlining the constructs used in this study for wild-type (lncRNA) or mutated (lncRNAS1-6M) lncRNA. (f) Western-blot analysis of E-cadherin, PNUTS and ZEB1 protein expression in A549 and NMuMG cells overexpressing wild-type (lncRNA) or mutated (lncRNAS1-6M) constructs of lncRNA-PNUTS and treated or not with synthetic miR-205 mimic or TGFβ for 3 days. TGFβ was used as a positive control. * PNUTS protein band. (g) Wound-healing migration assay of control (CTRL), lncRNA-PNUTS (lncRNA) or mutated lncRNA-PNUTS (lncRNAS1-6M) A549 and NMuMG cell models. Scale bar: 400µM (h) Modified Boyden chamber invasion assay of wild-type (lncRNA) or mutated (lncRNAS1-6M) lncRNA-PNUTS overexpressing A549 and NMuMG cells pre-treated +/− synthetic miR-205 mimic or TGFβ for 3 days. hnRNP E1 knockdown (E1KD) and TGFβ treated cells were used as a positive control. (Mean ± s.d., n= 3 independent experiments per condition, ANOVA followed by post-hoc Tukey's multiple comparisons test, *p<0.05; **p<0.01; ***p<0.001, NS, not significant). Source data are available in . (i) Confocal microscopy imaging of co-immunostaining of vimentin (green), E-cadherin (red) and merged with DAPI (blue) in A549 and NMuMG cells overexpressing wild-type (lncRNA) or mutated (lncRNAS1-6M) constructs of lncRNA-PNUTS and treated +/− synthetic miR-205 mimic or TGFβ for 3 days. Scale bar: 50µM. For all western-blots and RT-PCRs GAPDH was used as a loading control. Unprocessed original scans of blots are shown in

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Stable Transfection, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Control, shRNA, Modification, Boyden Chamber Assay, Microscopy, Knockdown, Western Blot, Construct, Expressing, Positive Control, Migration, Invasion Assay, Confocal Microscopy, Imaging, Immunostaining

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) Dual luciferase reporter assays to test the interaction between miR-205 and lncRNA-PNUTS (S3 to S6 region) by using a synthetic miR-205 mimic (+ miRNA 205) co-transfected with wild-type (LUC-lncRNA) or mutated (LUC-lncRNA-S3-S6M) constructs of lncRNA-PNUTS cloned into the 3'-UTR of the luciferase reporter gene. For each condition, assays were normalized to Renilla reporter gene expression. (mean ± s.d., n= 7 independent experiments per condition, two-tailed Student t test, ***p<0.001, NS, not significant). (b) Dual-Luciferase reporter assay of miR-205 bioavailability in A549 and NMuMG cells overexpressing wild-type (lncRNA) or mutated (lncRNAS1-6M) constructs of lncRNA-PNUTS. TGFβ treatment and hnRNP E1 knockdown (E1KD) were used as internal controls. For each condition, assays were normalized to Renilla reporter gene expression. (mean ± s.d., n= 4 independent experiments per condition, two-tailed Student t test, **p<0.01; ***p<0.001, NS, not significant). (c) The wild-type (REN-3'-UTR-ZEB1) 3'-UTR of ZEB1 cloned into the 3'-UTR of the Renilla gene was transfected in A549 and NMuMG cells overexpressing wild-type (lncRNA) or mutated (lncRNAS1-6M) constructs of lncRNA-PNUTS and treated +/− synthetic miR-205 mimic or TGFβ for 3 days. Mutated construct (REN-3'-UTR-ZEB1-mut(205)) for the miR-205 binding site located in the 3'-UTR of ZEB1 was also used. TGFβ was used as a positive control. For each condition, assays were normalized to Luciferase reporter gene expression. (mean ± s.d., n= 4 independent experiments per condition, two-tailed Student t test, **p<0.01; ***p<0.001, NS, not significant). (d) The wild-type (prom-CDH1-WT) proximal promoter of E-Cadherin driving the luciferase reporter gene expression was transfected in A549 cells overexpressing wild-type (lncRNA) or mutated (lncRNAS1-6M) constructs of lncRNA-PNUTS and treated or not with synthetic miR-205 mimics or TGFβ for 3 days. Mutated construct for both E2 Boxes 1 and 3 (Prom-CDH1-mEboxes) located on the promoter was also used. TGFβ was used as a positive control. For each condition, assays were normalized to Renilla reporter gene expression. (mean ± s.d., n= 4 independent experiments per condition, two-tailed Student t test, *p<0.05; **p<0.01; ***p<0.001, NS, not significant).

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Luciferase, Transfection, Construct, Clone Assay, Gene Expression, Two Tailed Test, Reporter Assay, Knockdown, Binding Assay, Positive Control

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: (a) Bright-field microscopy pictures of in vitro mammosphere/oncosphere formation assay in NMuMG and A549 cells overexpressing empty vector (CTRL), lncRNA-PNUTS (lncRNA) or mutated lncRNA-PNUTS (lncRNAS1-6M). Scale bar: 20µM (b) Absolute quantification of the sphere numbers obtained in (a). (mean ± s.d., n=5 independent experiments, two-tailed Student t test, ***p<0.001 NS, not significant). Source data are available in . (c) Number of tumor formed upon limiting dilution injection of control and lncRNA-PNUTS overexpressing MDA-468 cells. MDA-MB-468 cells were injected into the mammary fat pads of 6–8 week-old female mice in limiting dilution. TIC, tumor-initiating cells number was determined using ELDA software . Number of mice used for each condition is indicated. (d) Flow cytometry analysis of CD24/CD44 cell surface expression levels in the epithelial (CD44 − /CD24 + sorted cells) and mesenchymal (CD44 + /CD24 − sorted cells) HMLE subpopulations. (e) Cell morphology observed by phase-contrast microscopy. Scale bar: 50µM. (f) RT-PCR analysis of lncRNA-PNUTS expression level in mesenchymal and epithelial sorted HMLE cells. (g) Flow cytometry analysis of the CD24/CD44 cell surface expression levels in the epithelial (CD44 − /CD24 + sorted cells) HMLE subpopulation expressing empty vector (control) or overexpressing lncRNA-PNUTS. (h) Tumor weight of primary tumors obtained following mammary fat pad injection of MDA-231-LM2 expressing scrambled control (SCR) or lncRNA-PNUTS targeting shRNA (shRNA) in NOD/SCID mice. (mean ± s.d., n= 4 mice per condition, two-tailed Mann-Whitney test, p=0.08570). Source data are available in . (i) (Left) Histopathological analysis of paraffin-embedded lung serial sections of mice injected in the mammary fat pad with MDA-231-LM2 expressing scrambled or lncRNA-PNUTS targeting shRNA. Haematoxylin and Eosin (H&E) staining and immunostaining of Ki67 protein was performed in serial lung sections to identify macro- and micro-metastases. (Right) Photographs of primary tumors and of a representative lung collected for each condition. Scale bar: 500 µM

Article Snippet: NMuMG, A549, MCF7, CaCo-2, HMLE, MCF10a and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC), and the MDA231 progression model was graciously provided by Dr. Joan Massagué.

Techniques: Microscopy, In Vitro, Tube Formation Assay, Plasmid Preparation, Quantitative Proteomics, Two Tailed Test, Injection, Control, Software, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, MANN-WHITNEY, Staining, Immunostaining