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: 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: Neurobiology of disease

Article Title: HIV, Tat and Dopaminergic Transmission

doi: 10.1016/j.nbd.2017.04.015

Figure Lengend Snippet: Tat Models Referenced in Review

Article Snippet: Yuan , 2015 , Cell Line , PC12 and ATCC cells exposed to 500 nM Tat 1-86 , Radiolabeled uptake , Provides supporting evidence for data published in Midde et. al., 2015 in different cell line, again suggesting the importance of Y470 residue of DAT being critical to Tat-dependent alterations of DA transport..

Techniques: Variant Assay, Rnase Protection Assay, Blocking Assay, Sequencing, In Situ Hybridization, Cell Culture, Infection, Cell Counting, Produced, Transfection, Northern Blot, Labeling, Transactivation Assay, Clinical Proteomics, Membrane, Derivative Assay, Plasmid Preparation, Purification, Western Blot, Immunocytochemistry, Gene Expression, In Vitro, Expressing, Pulse Chase, Permeability, Chemotaxis Assay, Ligand Binding Assay, Staining, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Southern Blot, Injection, TUNEL Assay, Transgenic Assay, Binding Assay, Synthesized, Real-time Polymerase Chain Reaction, Flow Cytometry, Circular Dichroism, Incubation, Patch Clamp, Mass Spectrometry, Ab Array, Dot Blot, Silver Staining, Electrophoretic Mobility Shift Assay, Activity Assay, Microinjection, MTT Assay, Multiplex Assay, Activation Assay, SPR Assay, Animal Model, Phospho-proteomics, Immunoprecipitation, Luciferase, Sedimentation, Recombinant, Inhibition, Mutagenesis, Transmission Assay, Electron Microscopy, Immunohistochemistry, Immunodepletion, Control, Knock-Out, Conditioned Place Preference, Microscopy, Residue

Journal: Nature Communications

Article Title: CAV2-expressing nerves induce metabolic switch toward mitochondrial oxidative phosphorylation to promote cancer stemness

doi: 10.1038/s41467-025-66914-2

Figure Lengend Snippet: A Immunoblot analysis of Cav2 in TGs that were either cultured alone (day 0) or co‑cultured with SCC15 or MOC2 cells for 5 days. This experiment was independently repeated three times. B Immunofluorescence studies reveal an up-regulated Cav2 expression in TG neurons from B6.129(Cg)- Gt ( ROSA ) 26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J mice co-cultured with SCC15 or MOC2 cells. Results were consistent across images from 10 cells. Scale: 100 μm. C Subcellular location of Cav2 in the growth cone of TG neurons co-cultured with SCC15. Results were consistent across images from 10 cells. Scale: 100 μm. D Cav2 expression in non-neuronal cells within the TG does not undergo significant changes after co-culturing with SCC15. Results were consistent across images from 10 cells. Scale: 100 μm. E Transmission electron microscopy of PC12 cells solo- vs. co-cultured with SCC15, highlighting caveolae-like structures (50–100 nm). Scale: 500 nm. n = 5 biologically independent co-cultures/group. * p = 0.0492, Student’s t-test (two-sided). Data are mean ± s.d. F Representative images and quantitative analysis of caveolae-like structures in DRG cells. Scale: 1μm. n = 5 biologically independent co-cultures/group. n.s: p = 0.3349, Student’s t-test (two-sided). Data are mean ± s.d. G Immunofluorescence and analysis of Cav2 on DiI-labeled TG neurons from MOC2 xenografts versus sham. Sham controls received injections of tumor‑cell–free buffer (30 µL of DMEM/Matrigel at a 1:1 ratio). These images depict Cav2 expression primarily on the surface of certain TG neurons Scale: 40μm. n = 5 mice per group. ** p = 0.0096, Student’s t-test (two-sided). Data are mean ± s.d. H Cav2 nuclear expression in TG neurons from MOC2 xenografts versus sham. Scale: 100μm. n = 5 mice per group. n.s: p = 0.9502, Student’s t-test (two-sided). Data are mean ± s.d. I Quantitative analysis illustrating the prevalence of Cav2 surface expression on DiI + neurons in both MOC1 and sham conditions. n = 5 mice per group. * p = 0.475, Student’s t-test (two-sided). Data are mean ± s.d. J An illustrative diagram depicting the 4-NQO-induced murine HNSCC model. K , L Representative images and quantitative analysis of densities of both sensory nerves (Trpv1 + ) and Cav2 + nerves during various stages of 4-NQO-induced murine tumorigenesis. Scale: 50μm. n = 8 mice per group. * p = 0.0231, ** p = 0.0057(line 1 vs line 2), ** p = 0.0090 (line 2 vs line 3), **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. M Proportion of Cav2 + nerves among total sensory nerves. n = 8 mice per group. n.s: p = 0.2575; **** p < 0.0001, Student’s t-test(two-sided). Data are mean ± s.d.

Article Snippet: SCC15 human squamous carcinoma cells (ATCC, CRL-1623), PC-12 rat pheochromocytoma cells (National Collection of Authenticated Cell Cultures, TCR-9), and murine oral carcinoma cell lines MOC1 (BFN6021632) and MOC2 (BFN6021637) (BLUEFBIO) were used.

Techniques: Western Blot, Cell Culture, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Labeling

Journal: Nature Communications

Article Title: CAV2-expressing nerves induce metabolic switch toward mitochondrial oxidative phosphorylation to promote cancer stemness

doi: 10.1038/s41467-025-66914-2

Figure Lengend Snippet: A Left: Diagrammatic representation of the transwell chamber assay used to assess the migration of human HNSCC SCC15 cells or murine MOC2 cells towards murine TGs or DRGs. Middle: Representative images display SCC15 or MOC2 cells being attracted by Cav2 +/+ TGs, Cav2 +/- TGs, or Cav2 -/- TGs. Right: Statistical summary detailing the average number of HNSCC cells in each visual field. Scale: 200μm. n = 3 biologically independent co-cultures/group. **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. B Cancer cells were cultured in a Matrigel environment adjacent to TGs. Images present neural infiltration by SCC15 cells in association with TG from a heterozygous Cav2-deficient mouse (Cav2 +/- ), a homozygous deficient mouse (Cav2 -/- ), and their wild-type counterparts. Analysis encompassed nerve invasion index (α/β) and neurite length measurements. Scale: 750μm. n = 3 biologically independent co-cultures/group. Invasion index: * p = 0.0181, ** p = 0.0085, Outgrowth neurites: * p = 0.0476. Student’s t-test (two-sided). Data are mean ± s.d. C Fluorescence images of TG neurons post-co-culture with SCC15 cells or controls. The subsequent quantification evaluates both neuritogenesis and the length of the most extended axons, observed 72 h post co-culture. Scale: 100μm. First and fourth groups: n = 11 biologically independent co-cultures; second and third groups: n = 10. Length of longest axon: * p = 0.0150, Number of neural filaments: * p = 0.0301. Student’s t-test (two-sided). Data are mean ± s.d. D The percentage of PC12 cells, transfected with Cav1 or Cav2 plasmids, exhibiting neurite outgrowth of at least 10μm after 48 h of stimulation with 50 ng/mL NGF. n = 3 biologically independent cell cultures/group. ** p = 0.0016, *** p = 0.0010, Student’s t-test (two-sided). Data are mean ± s.d. E Western blot analysis of p75NTR levels in the lipid raft fraction before and after the addition of NGF (20 ng/mL for 1 h). Experiments were independently repeated three times. F Western blot analysis of the phosphorylation levels of RSK and CREB in control and Cav1 or Cav2-transfected PC12 cells under 20 ng/mL NGF stimulation at various time points. Experiments were independently repeated three times. TG trigeminal ganglion, DRG dorsal root ganglion, HNSCC head and neck squamous cell carcinoma, WT wild-type, HET heterozygous, KO knockout.

Article Snippet: SCC15 human squamous carcinoma cells (ATCC, CRL-1623), PC-12 rat pheochromocytoma cells (National Collection of Authenticated Cell Cultures, TCR-9), and murine oral carcinoma cell lines MOC1 (BFN6021632) and MOC2 (BFN6021637) (BLUEFBIO) were used.

Techniques: Transwell Chamber Assay, Migration, Cell Culture, Fluorescence, Co-Culture Assay, Transfection, Western Blot, Phospho-proteomics, Control, Knock-Out

Journal: Nature Communications

Article Title: CAV2-expressing nerves induce metabolic switch toward mitochondrial oxidative phosphorylation to promote cancer stemness

doi: 10.1038/s41467-025-66914-2

Figure Lengend Snippet: A Schematic overview of RNA sequencing performed on SCC15 cells harvested post 72-h co-culture with Cav2 -/- or wild-type mouse TGs. (B, C) GSEA profiling reveals significantly upregulated pathways in SCC15 cells co-cultured with Cav2 +/+ TGs over Cav2 -/- TGs. Two-sided permutation test. EMT: EPITHELIAL MESENCHYMAL TRANSITION; OXPHOS‌: OXIDATIVE PHOSPHORYLATION. D Evaluation of OCRs in SCC15 cells post 72-h co-culture with or without Cav2 +/+ TGs or Cav2 -/- TGs. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. n = 3 biologically independent co-cultures/group. Basal: ** p = 0.0012, *** p = 0.0007, FCCP: line 1 vs line 2: p = 0.0051; line 2 vs line 3: p = 0.0037. Student’s t-test (two-sided). Data are mean ± s.d. E MitoSOX Red staining and flow cytometry analysis of SCC15 cells after 72-h co-culture conditions. n = 3 biologically independent co-cultures/group. * p = 0.0132, Student’s t-test (two-sided). Data are mean ± s.d. F ROS-Glo determination of ROS levels. n = 3 biologically independent co-cultures/group. * p = 0.0236, ** p = 0.0024, Student’s t-test. Data are mean ± s.d. G SCC15 cells from different co-culture conditions stained with MitoTracker Red CMXRos and analyzed via flow cytometry. n = 3 biologically independent co-cultures/group. *** p = 0.0003, **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. H Transmission electron microscopy on SCC15 cells post co-culture conditions, with mitochondrial counts recorded. Scale: 2μm. n = 8 biologically independent co-cultures per group. ** p = 0.0030, *** p = 0.0005, Student’s t-test (two-sided). Data are mean ± s.d. I Mitochondrial DNA quantification in SCC15 cells using real-time qPCR and dedicated primer sets. n = 3 biologically independent co-cultures/group. line 1 vs line 2: p = 0.0088; line 2 vs line 3: p = 0.0047, Student’s t-test (two-sided). Data are mean ± s.d. J Kaplan-Meier survival plots comparing overall survival (OS) in CAV2 high and CAV2 low subgroups within both HIF1A high and HIF1A low HNSCC TCGA datasets. Two-sided log-rank test. HIF1A high group: n = 250 patients; HIF1A low group: n = 250 patients. TG trigeminal ganglion, NES normalized enrichment score, OCR oxygen consumption rate, ROS reactive oxygen species.

Article Snippet: SCC15 human squamous carcinoma cells (ATCC, CRL-1623), PC-12 rat pheochromocytoma cells (National Collection of Authenticated Cell Cultures, TCR-9), and murine oral carcinoma cell lines MOC1 (BFN6021632) and MOC2 (BFN6021637) (BLUEFBIO) were used.

Techniques: RNA Sequencing, Co-Culture Assay, Cell Culture, Phospho-proteomics, Staining, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Nature Communications

Article Title: CAV2-expressing nerves induce metabolic switch toward mitochondrial oxidative phosphorylation to promote cancer stemness

doi: 10.1038/s41467-025-66914-2

Figure Lengend Snippet: A ALDH1A1 enzymatic activity assessed via the ALDEFLUOR assay in SCC15 cells. ALDH1 + cells, isolated by flow cytometry, were subsequently evaluated for their OCRs. n = 3 biologically independent experiments per group. Data are mean ± s.d. B Flow cytometry of MitoSOX Red-stained SCC15 cells sorted as in ( A ). n = 3 biologically independent experiments per group. * p = 0.0295, Student’s t-test (two-sided). Data are mean ± s.d. C MitoTracker Red CMXRos staining followed by flow cytometric analysis. n = 3 biologically independent experiments per group. * p = 0.0302, Student’s t-test. Data are mean ± s.d. D GSEA reveals stemness gene set enrichment in SCC15 cells co-cultured with Cav2 +/+ TGs over Cav2 -/- TGs. Two-sided permutation test. E Representative images and quantification of CD44 + cells in human HNSCC tissues ( n = 7 niches from different patients). Scale: 100μm. * p = 0.0271, Student’s t-test (two-sided). Data are mean ± s.d. (F-G) The percentage of ALDH1 + cells of SCC15 ( F) and MOC2 ( G) cells post specified co-culture conditions. n = 3 biologically independent co-cultures/group. F : * p = 0.0487, ** p = 0.0019, G : * p = 0.0168, ** p = 0.0033, Student’s t-test (two-sided). Data are mean ± s.d. H Representative tumorspheres from SCC15 cells alone or co-cultured with Cav2 +/+ or Cav2 -/- TGs, with quantification of number and diameter. Scale: 200μm. n = 5 biologically independent co-cultures/group. * p = 0.0269, *** p = 0.0005, **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. I Cells treated with cisplatin, followed by flow cytometry for apoptotic cells. Four days later, cell viability was measured (CCK‑8 assay). n = 5 biologically independent experiments per group. ** p = 0.0086, **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. J MOC2 cells were orthotopically injected into C57BL/6 mice either alone or combined with Cav2 +/+ or Cav2 -/- TGs at a 1:1 volume ratio ( n = 8 per group). Tumor formation was evaluated after two weeks, with the incidence rates being subsequently reported. χ²-test (two-sided). K Representative multicolor immunohistochemical staining of Aldh1a1, CD44, and Bmi1 in the tongues of MOC2-engrafted wild-type versus Cav2 knockout mice. Cells positive for any one of these markers are defined as marker-positive cells. Scale: 500μm. n = 3 mice per group. **** p < 0.0001, Student’s t-test (two-sided). Data are mean ± s.d. OCR oxygen consumption rate, TG trigeminal ganglion, NES normalized enrichment score.

Article Snippet: SCC15 human squamous carcinoma cells (ATCC, CRL-1623), PC-12 rat pheochromocytoma cells (National Collection of Authenticated Cell Cultures, TCR-9), and murine oral carcinoma cell lines MOC1 (BFN6021632) and MOC2 (BFN6021637) (BLUEFBIO) were used.

Techniques: Activity Assay, Isolation, Flow Cytometry, Staining, Cell Culture, Co-Culture Assay, CCK-8 Assay, Injection, Immunohistochemical staining, Knock-Out, Marker

Journal: Nature Communications

Article Title: CAV2-expressing nerves induce metabolic switch toward mitochondrial oxidative phosphorylation to promote cancer stemness

doi: 10.1038/s41467-025-66914-2

Figure Lengend Snippet: A Cav2 is expressed in the Golgi apparatus of neurons within the trigeminal ganglia. Representative images are shown. Results were consistent across images from 10 cells. Scale: 50 μm. B Cav2 is also localized to the Golgi apparatus in non-neuronal cells of the trigeminal ganglia. Results were consistent across images from 10 cells. Representative images are shown. Scale: 50 μm. C After co-culture with SCC15 cells for 2 h, a subset of neurons exhibited Cav2 redistribution along the axons. Results were consistent across images from 5 cells. Scale: 20 μm. D Trigeminal ganglia from wild-type mice were cultured alone or co-cultured with SCC15 tumor cells for 48 h, followed by a 6-h period of independent culture in fresh medium. The protein concentration in the supernatant was subsequently measured. n = 3 biologically independent experiments per group. ** p = 0.0057, Student’s t-test (two-sided). Data are mean ± s.d. E Trigeminal ganglia from Cav2 +/+ or Cav2 -/- mice were co-cultured with SCC15 tumor cells for 48 h, followed by a 6-h independent culture in fresh medium. The protein concentration in the supernatant was measured. n = 3 biologically independent experiments per group. * p = 0.0330, Student’s t-test (two-sided). Data are mean ± s.d. F Mass spectrometry analysis was performed on supernatants collected from Cav2 +/+ and Cav2 -/- trigeminal ganglia co-cultured with SCC15 cells for 48 h, followed by a 6-h independent culture. The figure presents only those proteins with a fold change (FC) ≥ 2 or ≤1/2 and p < 0.05. n = 3 biologically independent experiments per group. Student’s t-test (two-sided). WT wild-type, TG trigeminal ganglion.

Article Snippet: SCC15 human squamous carcinoma cells (ATCC, CRL-1623), PC-12 rat pheochromocytoma cells (National Collection of Authenticated Cell Cultures, TCR-9), and murine oral carcinoma cell lines MOC1 (BFN6021632) and MOC2 (BFN6021637) (BLUEFBIO) were used.

Techniques: Co-Culture Assay, Cell Culture, Protein Concentration, Mass Spectrometry

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: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: (A) Normal human female karyotype prepared from WT and LRRK2 G2019S iPSCs. Cytogenetic analysis was performed on 20 G-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 tri-lineage 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. (C) 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

Techniques: Immunostaining, Expressing, Staining, Generated, Quantitative RT-PCR, Gene Expression, Control

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: (A) Astrocytes were prepared from iPSCs carrying the 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 up-regulated genes and a 0.7-fold threshold for down-regulated 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 to 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 up-regulated 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

Techniques: Mutagenesis, Control, Derivative Assay, Expressing, Marker, Sequencing, Transformation Assay, Quantitative Proteomics, Labeling, Gene Expression, RNA Sequencing, Biomarker Discovery, Two Tailed Test, Standard Deviation

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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, 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.

Article Snippet: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: (A) Transmission electron microscopy (TEM) images of multi vesicular bodies (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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: (A,B) Quantification of CD63 gold particles shown as the number of particles per µm 2 MVB (A) and relative frequency (B) in WT vs LRRK2 G2019S astrocytes. Error bars represent mean + SEM. (C) 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 ACM using an exosome antibody array. Each circle on the membrane represents a pre-printed antibody spot marker of exosome or cellular contaminant, and the table details the name of each antibody marker spotted on the membrane. (E) Cryo-EM images of astrocyte-derived EVs that display an unusual morphology. (H) Quantification of the number of CD63 + EVs secreted in WT and LRRK2 G2019S ACM by ELISA, using ELISA standards calibrated by 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: (A) TEM images demonstrate that 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 x 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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 (> 3,000 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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 (> 3,300 MVBs) were analyzed for each experimental condition. Statistical analysis was performed using a Mann-Whitney test (**** p<0.0001).

Article Snippet: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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 alpha-synuclein (αSyn) in WT and LRRK2 G2019S 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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 iPSC-based model system .

Article Snippet: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

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

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

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 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 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: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

Techniques: Cell Culture, Two Tailed Test

Journal: bioRxiv

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: 10.1101/2020.07.02.178574

Figure Lengend Snippet: Gene ontology analysis of isogenic (Ai) and non-isogenic (Aii) LRRK2 G2019S vs WT iPSC-derived astrocytes showing down-regulated 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 down-regulated 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.

Article Snippet: Primary cultures were prepared via dissection of P1 to P3 mouse pups obtained from C57BL/6NTac WT mice or constitutive LRRK2 G2019S knock-in mice generated and maintained on the C57BL/6NTac background ( ) (Taconic Biosciences, NY) using methods approved by the Janelia Institutional Animal Care and Use Committee.

Techniques: Derivative Assay, RNA Sequencing