Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet:

Article Snippet: SMM-293 TII serum-free media , SinoBiological , M293TI.

Techniques: Binding Assay, Recombinant, Transfection, Luciferase, Bradford Protein Assay, Plasmid Preparation, Software, Single Particle, Microscopy

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 1. Atheroprotective shear stress (SS) to endothelial cells (ECs) suppresses microRNA-126 (miR-126) in the cocultured smooth muscle cells (SMCs). A, Schematic diagrams of EC/SMC coculture and flow system. B, Human umbilical vein ECs and human umbilical artery SMCs were seeded on the lower and upper sides of a membrane, respectively (EC/SMC). Controls had SMCs but no ECs on the other side (Ø/SMC). The temporal expression levels of primary (pri–miR-126) and mature (miR-126) forms of miR-126 were determined by quantitative reverse transcription–polymerase chain reaction. *P<0.05 vs Ø/SMC 0-hour static. C, The levels of miR-126 in the 24-hour static or sheared media. *P<0.05 vs static control. D, The levels of miR-126 in SMCs incubated with medium 199 supplemented with 2% fetal bovine serum (control [CL] media) or conditioned medium from static or sheared ECs (24-hour shearing) (EC-CM). *P<0.05 vs CL media. E, miR-126 expression in EC monoculture or SMC coculture for 24 hours. F, miR-126 expression in ECs exposed to laminar SS (LSS) for 24 hours. G, The levels of pri–miR-126 and miR-126 in EC coculture with SMCs under 24–hour static or LSS conditions.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Membrane, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Incubation

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 2. Endothelial cells (ECs) and shear stress (SS) regulate expressions of microRNA-126 (miR-126) targets in smooth muscle cells (SMCs). A, The mRNA levels of forkhead box O3 (FOXO3), B-cell lymphoma 2 (BCL2), and insulin receptor substrate 1 (IRS1) in Argonaute2 (Ago2) immunocomplexes in SMCs 48 hours after transfection with microRNA-126 mimics (PRE126) or anti–microRNA-126 inhibitor (AM126). *P<0.05 vs cells transfected with negative controls (negative control mimics [PREC] or negative control inhibitor [AMC]). B, The relative luciferase levels in SMCs cotransfected with PRE126 (left), AM126 (right), or the negative control and luciferase reporter plasmids: empty vector only, wild-type (WT) 3′-untranslated region (3′UTR) of FOXO3, BCL2, or IRS1 and mutants of 3′UTR of FOXO3, BCL2, or IRS1. C, The relative luciferase levels in SMCs transfected with the indicated luciferase reporter plasmids and then incubated with control media or EC-conditioned media (CM). *P<0.05 vs cells transfected with empty vector. #P<0.05 vs cells transfected with plasmids harboring the WT 3′UTRs. D, The protein levels of target genes in SMCs monocultured (Ø/SMC) or cocultured with ECs (EC/SMC) kept under static condition or exposed to laminar shear stress (LSS). Images are representative of triplicate experiments with similar results.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Luciferase, Plasmid Preparation, Incubation, Control

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 3. Endothelial cells (ECs) transfer exogenous microRNA-126 (miR-126) to smooth muscle cells (SMCs). SMCs were incubated with conditioned media (CM) derived from ECs transfected with biotinylated synthetic miR-126 (B-miR-126) or no-miR mock control (Mock) for 3 hours. A, The levels of B-miR-126 in ECs or the EC-CM were determined by pull-down experiments with streptavidin- agarose beads followed by quantitative reverse transcription–polymerase chain reaction, (B) and B-miR-126 in ECs and SMCs was detected with QDot-605–streptavidin conjugates. *P<0.05 vs mock control. C and D, SMCs were cocultured with ECs transfected with B-miR-126 or mock control for 24 hours; the levels of B-miR-126 in SMCs were determined by Northern blot (C) or pull-down experiments with streptavidin-agarose beads (D). *P<0.05 vs mock control.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Incubation, Derivative Assay, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction, Northern Blot

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 4. Functional microRNA-126 (miR-126) transmission from endothelial cells (ECs) to smooth muscle cells (SMCs) is independent of vesicles. A, The levels of miR-126 in SMCs incubated with control (CL) media or the EC-conditioned media (CM) treated with DNase I, RNase, or proteinase K (PK) for 3 hours. *P<0.05 vs CL media. B, The levels of miR-126 in ECs transfected with control small interfering (si)RNA; siCL) or siRNA against Drosha (siDROSHA), Dicer (siDICER1) or Argonaute2 (siEIF2C2) or in SMCs incubated with CL media or EC-CM derived from those transfected ECs. C, The levels of miR-126 in SMCs incubated EC-CM derived from ECs transfected with anti–miR-126 inhibitor anti–miR-126 inhibitor (AM126), miR-126 mimics (PRE126), or the negative controls. *P<0.05 vs siCL or CL media. #P<0.05 vs the negative control inhibitor (AMC) or negative control mimics (PREC) EC-CM. D, The levels of miR-126 in SMCs cocultured with lung ECs isolated from wild-type (WT) or miR-126 knockout mice (KO). *P<0.05 vs Ø/SMC (SMC monocultured). EC-CM was ultracentrifuged to fractionate the components in spin-down pellets (Pellet; which was reconstituted with M199), or the remaining supernatant (Super). The levels of miR-126 in (E) the fractionated EC-CM or in (F) SMCs incubated with total or fractionated EC-CM for 3 hours. G, The transcript levels or (H), protein levels of indicated genes in SMCs incubated with the fractioned EC-CM. Images are representative of triplicates with similar results. *P<0.05 vs CL media.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Functional Assay, Transmission Assay, Incubation, Control, Transfection, Derivative Assay, Negative Control, Isolation, Knock-Out

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 5. Argonaute2 (Ago2) associates with microRNA-126 (miR-126) and facilitates its transmission. A, The levels of miR-126 in endothelial cell–conditioned media (EC-CM) immunocomplexes of Ago2 or Ago1 or control immunoglobin G (IgG). *P<0.05 vs IgG control. B, Smooth muscle cells (SMCs) were cocultured with bovine aortic ECs transfected with a plasmid encoding the eGPF-Ago2. The enhanced green fluorescent protein-Ago2 proteins were visualized in live ECs or SMCs by microscopy. C, The protection of miR-126 with preincubation of bovine serum albumin (BSA) or recombinant human Ago2 protein (rAgo2) for 30 minutes after RNase A digestion for 15 minutes. *P<0.05 vs miR-126 only. D, The enrichment of miR-126 in SMCs treated with miR-126 preincubated with BSA or rAgo2 for 3 hours. *P<0.05 vs miRs only. E, The levels of Ago2 in ECs (left) or EC-CM (right) under static or laminar shear stress (LSS) conditions for 24 hours. F, The levels of miR-126 in Ago2 immunocomplex in EC-CM under static of LSS conditions. Images are representative of triplicates with similar results. Semiquantification results are shown in the lower panels. *P<0.05 vs static.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Transmission Assay, Control, Transfection, Plasmid Preparation, Microscopy, Recombinant, Shear

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 6. Shear stress (SS)–imposed endothelial cells (ECs) modulate smooth muscle cell (SMC) turnover through microRNA-126 (miR-126). SMCs were monocultured or cocultured with (1) ECs kept under static condition or exposed to laminar shear stress (LSS) for 24 hours; or (2) ECs transfected with negative control inhibitor (AMC) or anti–miR-126 inhibitor (AM126). A, Proliferating cell nuclear antigen (PCNA) in the SMCs was detected by immunostaining. B, Protein levels of PCNA, cyclinA, and p21 in SMCs were assayed by Western blot. C and D, SMC proliferation and apoptosis analyzed for propidium iodide–stained DNA content and Annexin V–stained membrane flip, respectively. *P<0.05 vs Ø/SMC (SMC monocultured). #P<0.05 vs Static EC-SMC or AMC EC-SMC.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Immunostaining, Western Blot, Staining, Membrane

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 7. Depletion of microRNA-126 (miR-126) in mice inhibits smooth muscle cell (SMC) proliferation and neointimal formation in arteries after cessation of blood flow. A, The miR-126 expression levels in mouse carotid arteries with or without ligation. Vascular endothelial cells (ECs) were denuded before RNA isolation. *P<0.05 vs unligated control. B, In situ hybridization of miR-126 in ligated or unligated carotid arteries from miR-126 wild-type (WT) mice. Yellow arrows, elastic lamina (EL). C, Representative images of modified Verhoeff-Van Gieson–stained ligated or unligated carotid arteries from WT or miR-126 knockout (KO) mice. Arrows indicate the thickened neointima. D and E, Representative images of immunofluorescent-stained proliferating (proliferating cell nuclear antigen [PCNA]) SMC (smooth muscle-α actin) in ligated or unligated carotid arteries from WT and KO mice. F, Representative images of modified Verhoeff-Van Gieson–stained ligated carotid arteries from WT or KO mice treated with control (CL) media or EC-conditioned media (EC-CM); or with local delivery of CL oligo or miR-126 mimics (PRE126). Arrows indicate thickened neointima. G, In situ hybridization of miR-126 in ligated carotid arteries from KO mice with treatment of EC-CM, miR-126, or the respective controls.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Expressing, Ligation, Isolation, Control, In Situ Hybridization, Modification, Staining, Knock-Out

Journal: Scientific Reports

Article Title: Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

doi: 10.1038/srep38792

Figure Lengend Snippet: ( A) The EphB4 receptor is present at high to moderate levels in human HNSCC cells compared to the normal oral keratinocyte (NOK) cells as detected by Western blotting. (B) EphB4 expression is reduced upon transfection with the EphB4-targeting siRNAs 1 or 2 compared to the control non-specific siRNA (NS-siRNA). (C–E) Reduction in survival fractions in HNSCC cells is observed after transfection with the EphB4-targeting siRNA versus the control NS-siRNA (25-50 nM) in Cal27 (C) , MSK-921 (D) , and Fadu (E) cells as determined by clonogenic assay. Each clonogenic assay was repeated atleast three times. Representative survival plots are shown for each cell line. The survival plot for the MSK-921 cells was generated using 0-6 Gy dose of ionizing radiation because 8 Gy dose did not yield viable colonies. Data shown represent mean ± standard deviation. *p < 0.05; **p < 0.01.

Article Snippet: HNSCC cells were transfected in serum-free, antibiotic-free growth medium using Mirus TransIT-TKO Transfection Reagent (Madison, WI, USA), according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Clonogenic Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

doi: 10.1038/srep38792

Figure Lengend Snippet: This table lists the survival fractions at 2 Gy dose of ionizing radiation (SF2) for each individual cell line comparing effects of the EphB4-siRNA transfection to those of the NS-siRNA transfection.

Article Snippet: HNSCC cells were transfected in serum-free, antibiotic-free growth medium using Mirus TransIT-TKO Transfection Reagent (Madison, WI, USA), according to the manufacturer’s instructions.

Techniques: Transfection

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: Tenascin C contributes to NRP-1 associated migration. a. Dual immunofluorescence staining (40× magnification scale bar 10 μm) of NRP-1 and TNC on BT-474 and BT-474 NRP-1 cells indicates their colocalization in the cytoplasm. Treatment of BT-474 NRP-1 cells with TNC targeted siRNA molecules, b , reduced TNC gene expression, c , reduced migratory capacity and d , downregulated NRP-1 and vimentin expression. (TNC protein was not detected on western blot due to the lack of specific antibody for this application.) The gene expression fold change was measured by comparing the basal levels detected in the empty plasmid transfected BT-474 or in the case of the siRNA experiment, to the control siRNA treated BT-474 and normalized to β-Actin and GUSB reference gene expression. Wound healing assay images (panel d, 5× magnification, scale bar 500 μm) taken on day 0 and day 2 after siRNA transfection. Graphs represent the mean ± SEM of three independent experiments. Statistical analysis using independent samples t-test, p-value < 0.05 considered as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Gene Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Wound Healing Assay

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: NRP-1 overexpression activates integrin β3 and TNFR2 pathways. Representative western blot images of protein lysates from untransfected BT-474, BT-474 NRP-1 and empty vector control cells blotted with indicated antibodies involved in a. Integrin signaling, and downstream signaling targets FAK, Akt, GSK3-β and NF-kB b. siRNA-mediated TNC downregulation decreased phosphorylation of FAK and Akt-473. c. Blots show levels of tumor necrosis factor receptors (TNFRs). GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control, Phospho-proteomics, Expressing

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: A and B. Surface expression or secretion of galectins. CFSE-labeled TH17 trophozoites (green) were co-incubated with h VECs (moi 1:3) were sampled at intervals for the detection of galectin-1 (red) and galectin-3 (cyan) by IFA (A), while these galectins in the conditioned medium were detected by ELISA (B). PI, post-infection. Adherent trophozoites in A are indicated by white triangles. C, D, and E. T1 or TH17 trophozoites were incubated in fresh or conditioned KSFM (D) from h VECs cultures for 30 min. Cells were fixed for detection of galectin-1 or -3 by IFA without permeation for (C) or with permeation for (D and E) before immune detection. E. IFA co-staining with anti-galectin-3 antibody and LysoTracker. F and G. T1 or TH17 trophozoites were incubated with FITC-conjugated recombinant galectin-1 or galectin-3 and analyzed by flow cytometry (F) or fluorescence microscopy (G). The bar graph is shown by mean ± SD. All assays were repeated three times and the representative data are shown here. Differences were statistically analyzed by Student’s t-test, with P <0.01(**) and P <0.05(*).

Article Snippet: Immortalized human vaginal epithelial cells ( h VECs, VK2/E6E7, ATCC CRL-2616) and HeLa cells were respectively maintained in semi-defined keratinocyte serum-free medium (KSFM) (GIBCO-BRL 17005–042, ThermoFisher Scientific) and DMEM high glucose medium (12100046, ThermoFisher Scientific) with 10% fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Expressing, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Staining, Recombinant, Flow Cytometry, Fluorescence, Microscopy

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: The tract of a single TH17 or T1 trophozoite migrating on a glass slide for 1 min is depicted in A. The migratory paths from ~50 trophozoites are shown in B, with the average track displacement length (TDL). The average velocities with standard deviations were statistically analyzed by Student’s t-tests as shown in C. The relative velocity of T1 in D. or TH17 in E., the trophozoites cultured in KSFM or with h VECs over 15 min was normalized by the average velocity of those in 1 min post-incubation in KSFM. The assays were repeated three times. For D. and E., the statistical analysis was measured by Bonferroni post hoc tests (n = 10 to 30 for each group). The error bars represent standard deviations, P <0.01(**) and P <0.05(*).

Article Snippet: Immortalized human vaginal epithelial cells ( h VECs, VK2/E6E7, ATCC CRL-2616) and HeLa cells were respectively maintained in semi-defined keratinocyte serum-free medium (KSFM) (GIBCO-BRL 17005–042, ThermoFisher Scientific) and DMEM high glucose medium (12100046, ThermoFisher Scientific) with 10% fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Cell Culture, Incubation

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 3 Effect of grape skin fermentation solution on cell growth. (A) Effect of treatment with grape skin fermentation solution for 48 hours on 293T cell growth and viability. (B) Effect of treatment with grape skin fermentation solution for 48 hours on HepG2 cell growth and viability (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques:

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 4 Effect of treatment grape skin fermentation solution for 48 hours on 293T cell morphology (200) (CF: the fermentation solution fer- mented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques:

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 6 SOD, GSH, CAT, and GSH-Px mRNA expressions in 293T cells. a–eMean values with different letters in the same column differed significantly (P < 0.05) by Duncan's multiple range test. Values presented are the means standard deviation (N ¼ 3/group) (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques: Standard Deviation

Journal: RSC Advances

Article Title: Grape skin fermentation by Lactobacillus fermentum CQPC04 has anti-oxidative effects on human embryonic kidney cells and apoptosis-promoting effects on human hepatoma cells

doi: 10.1039/c9ra09863a

Figure Lengend Snippet: Fig. 7 SOD, GSH, CAT, and GSH-Px protein expression in 293T cells. Values presented are the means standard deviation (N ¼ 3/group) (CF: the fermentation solution fermented by LF-CQPC04; BF: the fermentation solution fermented by Lactobacillus delbrueckii subsp. Bulgaricus; and WE: the solution extracted by ethanol) (* stands P < 0.05, ** stands 0.05 < P < 0.01, **** stands 0.01 < P < 0.001, **** stands 0.001 < P < 0.0001).

Article Snippet: The 293T cells and human hepatoma (HepG2) cells (the HepG2 cell line was purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China)) were seeded in 96-well plates at 1 106 and cultured in a constant-temperature incubator at 37 C and 5% CO2 for 24 h with DMEM (H) and DMEM (L) (Solarbio Life Sciences, Beijing, China).

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: The Amyloid Clearance Defect in ApoE4 Astrocytes is Corrected by Epigenetic Restoration of NHE6

doi: 10.1101/243097

Figure Lengend Snippet: A. Fluorescent-based assay to monitor clearance of Aβ peptides by astrocytes. B. Representative micrographs (left) and quantification (right) of ApoE3 and ApoE4 astrocytes subjected to 24h of Aβ uptake. Fluorescence intensity and exposure settings were kept constant. Following background subtraction, fluorescence signal from cell associated Aβ was reduced by 78% in ApoE4 astrocytes, relative to ApoE3 (****p=9.6×10 −71 ; n =100/condition; Student’s t-test). C. Representative fluorescence-activated cell sorting (FACS) histograms (left) demonstrating Aβ internalization by ApoE3 (top; orange) and ApoE4 astrocytes (bottom; grey) at 1h, 4h, and 16h; 10,000 cells/experimental condition. x-axis depicts Aβ clearance in logarithmic scale and vertical dashed line represents median fluorescence intensity. Quantification (right) of biological triplicate measurements of Aβ clearance from FACS analysis of ApoE3 and ApoE4 cells. Note significantly lower cell-associated Aβ in ApoE4 relative to ApoE3 at all time points (53% lower, 1 h; 59% lower, 4 h; 65% lower, 16 h; ****p<0.0001; n =3; Student’s t-test). D. Representative FACS histograms (left) and quantification of mean fluorescence intensity of biological triplicates (right) demonstrating TFN uptake following 60 minutes of endocytosis by ApoE3 (green) and ApoE4 (grey) astrocytes (~1.5-fold higher; ****p=6.8×10 −5 ; n =3; Student’s t-test). x-axis of the FACS histogram depicts TFN uptake in logarithmic scale and vertical dashed line represents median fluorescence intensity. E. ApoE3 and ApoE4 astrocytes were incubated with fluorescent transferrin (TFN) for 1h, to compare steady-state TFN uptake by confocal microscopy. Fluorescence intensity and exposure settings were kept constant. Representative images are shown (left) and mean fluorescence ± s.e. was plotted (right). Following background subtraction, fluorescence signal was increased by ~2-fold in ApoE4 astrocytes, relative to ApoE3 (****p=3.5×10 −32 ; n =100/condition; Student’s t-test). F. Quantification of mean fluorescence intensity of biological triplicates demonstrating dextran uptake by ApoE3 and ApoE4 astrocytes (p=0.870; n =3; Student’s t-test). See related Supplementary .

Article Snippet: Briefly, cells were rinsed and incubated in serum-free medium for 30min, to remove residual transferrin and then incubated with Alexa Fluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (100 μg/ml) at 37°C for 60min.

Techniques: Fluorescence, FACS, Incubation, Confocal Microscopy

Journal: bioRxiv

Article Title: The Amyloid Clearance Defect in ApoE4 Astrocytes is Corrected by Epigenetic Restoration of NHE6

doi: 10.1101/243097

Figure Lengend Snippet: (A-B) Representative micrographs (A) and quantification using Pearson correlation (B) determining fractional colocalization of Aβ (red) with transferrin (TFN) (green) in DAPI-(blue) stained ApoE3 astrocytes, following 60 minutes of uptake. Note poor colocalization, as evident in the merge and orthogonal slices (Z) with fewer yellow puncta (Pearson’s correlation: 0.16±0.09; n =45), suggesting that a significant pool of internalized Aβ escapes recycling endosomes. Representative micrographs (C) and quantification using Pearson correlation (D) determining fractional colocalization of Aβ (red) with different endosomal-lysosomal compartment markers (green) in DAPI-(blue) stained ApoE3 astrocytes following 12h of uptake. Colocalization is evident in the merge and orthogonal slices (Z) as yellow puncta. (E-F) Time course of Aβ accumulation in ApoE3 and ApoE4 astrocytes by confocal microscopy. Representative images are shown (E) and mean fluorescence ± s.e. was plotted (F). ApoE4 cells show a reduced rate and total Aβ accumulation, relative to ApoE3 astrocytes (****p<0.0001; Student’s t-test). Scale bars, 10μm.

Article Snippet: Briefly, cells were rinsed and incubated in serum-free medium for 30min, to remove residual transferrin and then incubated with Alexa Fluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (100 μg/ml) at 37°C for 60min.

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: bioRxiv

Article Title: The Amyloid Clearance Defect in ApoE4 Astrocytes is Corrected by Epigenetic Restoration of NHE6

doi: 10.1101/243097

Figure Lengend Snippet: A. Compartment-specific, ratiometric, pH-sensitive fluorescence reporters to probe ApoE-isotype dependent differences in endosomal, lysosomal and cytoplasmic pH. Endosomal pH was measured by incubations with pH-sensitive FITC-Transferrin (TFN-FITC) together with pH non-sensitive Alexafluor 633-Transferrin (TFN-633). Lysosomal pH was measured by incubations with pH-sensitive pHrodo-green-Dextran (DXN-pHrodo green) together with pH non-sensitive Alexa Fluor 647-Dextran (DXN-647). Cytoplasmic pH was measured ratiometrically using pH-sensitive green and pH nonsensitive red fluorescence of BCECF dye. B. Endosomal pH in ApoE4 astrocytes was strongly reduced by ~0.84 pH unit, relative to ApoE3 (**p=0.0037; n=3 ; Student’s t-test). C. Lysosomal pH was elevated by >1 pH unit in ApoE4 astrocytes (****p=0.0009; n=3; Student’s t-test). D. Cytoplasmic pH showed no significant differences between ApoE3 and ApoE4 astrocytes (p=0.2904; n=3; Student’s t-test). E. Monensin treatment (50μM for 1h) corrected hyperacidic endosomal pH in ApoE4 astrocytes, relative to vehicle treatment (****p=0.0005; n=3; Student’s t-test). F. Quantitation of Aβ clearance from FACS analysis of 10,000 cells in biological triplicates confirmed restoration of Aβ clearance in ApoE4 astrocytes to ApoE3 levels with monensin treatment (****p=6.5×10 −7 ; n =3; Student’s t-test). G. Representative micrographs (left) and quantification (right) showing ~2.9-fold increase in cell-associated Aβ in ApoE4 astrocytes with monensin treatment (****p=2.4×10 −20 ; n =50; Student’s t-test). Scale bars, 10μm.

Article Snippet: Briefly, cells were rinsed and incubated in serum-free medium for 30min, to remove residual transferrin and then incubated with Alexa Fluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (100 μg/ml) at 37°C for 60min.

Techniques: Fluorescence, Quantitation Assay

Journal: bioRxiv

Article Title: The Amyloid Clearance Defect in ApoE4 Astrocytes is Corrected by Epigenetic Restoration of NHE6

doi: 10.1101/243097

Figure Lengend Snippet: A. Gene ontology (GO) analysis of top-100 downregulated genes in post-mortem AD brains obtained as described under “Experimental Methods.” Enrichment scores and p-values for top-5 GO biological process were shown. Note that genes involved in hydrogen ion transmembrane transport, including NHE6 and V-ATPase subunits exhibited highest enrichment scores. B. Quantitative PCR (qPCR) analysis of NHE9 transcript reveled significantly lower expression in ApoE4 astrocytes, relative to ApoE3 (~70% lower; ****p=2.7×10 −5 ; n =3; Student’s t-test). No changes in mRNA levels were observed for the closely related plasma membrane NHE1 isoform (p=0.946; n =3; Student’s t-test). Significantly lower mRNA levels for lysosomal V-ATPase V0a1 subunit in ApoE4 astrocytes, relative to ApoE3 (~67% lower; ****p=9.7×10 −5 ; n =3; Student’s t-test). C. Consistent with clinical reports of microcephaly in Christianson syndrome patients, 7-month old hemizygous NHE6 KO mice showed significantly lower brain weight, relative to wild-type mice. D. Lentiviral vector mediated expression of NHE6 in ApoE4 astrocytes with low endogenous NHE6 levels results in alkalization of endosomal pH. E. Representative micrographs (left) and quantification using Pearson correlation (right) determining fractional colocalization of NHE9 (green) with transferrin (TFN) (red) in DAPI-(blue) stained ApoE4 astrocytes following 60 minutes of uptake. Note prominent endosomal colocalization of NHE9 as evident in the merge and orthogonal slices (Z) as yellow puncta (Pearson’s correlation: 0.74±0.10; n =40). F. Representative FACS histograms (left) and quantification of mean fluorescence intensity of biological triplicates (right) demonstrating no difference in Aβ internalization between ApoE4 (red) and ApoE4 astrocytes with NHE9-GFP expression (green). x-axis depicts Aβ clearance in logarithmic scale and vertical dashed line represents median fluorescence intensity (p=0.155; n =3; Student’s t-test). (G-H) Representative immunofluorescence images of permeabilized, fixed ApoE4 astrocytes expressing NHE6-GFP showing overlap of NHE6 (green) with red-labeled EEA1 (G) and LRP1 (H). Colocalization is evident in the merge and orthogonal slices (Z) as yellow puncta. I. Representative blots showing surface (left) and total (right) LRP1 protein levels with NHE6-GFP expression (detected using anti-GFP antibody) relative to empty vector transfection. As loading control, surface biotinylated proteins were visualized with HRP-streptavidin and by probing against tubulin. GAPDH was used as a loading control for western analysis of total LRP1 levels. Quantification (extreme right) of blots showed robust ~5.7-fold higher surface LRP1 levels in ApoE4 cells with restored NHE6 expression, compared to transfection with empty vector (**p=0.002; n =3; Student’s t-test). No concomitant changes in total LRP1 levels (p=0.390; n =3; Student’s t-test) suggesting that increased surface LRP1 was due to posttranslational redistribution of the existing cellular LRP1 pool. J. qPCR analysis confirmed no concomitant changes in LRP1 transcript with lentiviral transduction of NHE6-GFP, relative to empty vector control (p=0.797; n =3; Student’s t-test). Scale bars, 10μm.

Article Snippet: Briefly, cells were rinsed and incubated in serum-free medium for 30min, to remove residual transferrin and then incubated with Alexa Fluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (100 μg/ml) at 37°C for 60min.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Staining, Fluorescence, Immunofluorescence, Labeling, Transfection, Western Blot, Transduction

Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A) Gene ontology enrichment of genes that are significantly upregulated in A20 ZF7 versus WT intestines by bulk RNAseq. Red bars highlight categories related to TH17 differentiation. Blue bars highlight categories related to cellular proliferation. (B) qPCR analyses of Il17a and Il22 expression from intact small intestine (relative to Actb ). (C) Volcano plot of all annotated UCSC Refseq genes from bulk RNAseq analyses of A20 ZF7 versus WT small intestines. Horizontal dashed line indicates adjusted p-value (FDR) of 0.01. (D) Representative immunohistochemical analyses of CD4 expression in WT and A20 ZF7 small intestines. Data are representative of 3 mice from each genotype. Bar, 100 microns. (E) Flow cytometry of small intestinal lamina propria cells from WT and A20 ZF7 mice. Data shown as mean + SEM. Statistics calculated using unpaired two-tailed Student t-test with Welch correction. *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, Immunohistochemical staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A, B) UMAP clusters of scRNAseq analyses of SILP from WT and A20 ZF7 mice. (C) Relative proportions of cell subsets in WT and A20 ZF7 SILP. TH17 subset includes both proliferative (mustard yellow) and non-proliferative (sky blue) compartments. (D) Projection of Il17a and Il22 expression onto UMAP clusters shown in panels A and B. (E) Violin plots of Il17a and Il22 expression in TH17 cells from indicated genotypes of mice. Statistics calculated using unpaired two-tailed Wilcoxon rank sum test. *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A) Flow cytometric analysis of RORγt expression in in vitro differentiated TH17 cells from WT and A20 ZF7 mice. (B) qPCR analyses of Il17a and Il22 expression in cells generated as in (A) above. “TH17” indicates TH17 differentiation conditions. “FICZ” indicates treatment with the AHR agonist FICZ. Note increased IL-22 expression in absence of supplementary treatment with PMA/ionomycin. (C) ELISA of IL-22 secretion from cells generated as in (A). (D) ATAC-seq of genomic loci at/near the Il22 locus in cells generated as in (A) above. Note increased DNA accessibility across Il22 gene in enlarged plot (locus g). (E) Chromatin IP of acetylated H3K27 at indicated Il22 loci (a,b,c in (D) above) in cells generated as in (A) above. Note increased H3K27 acetylation at locus b in A20 ZF7 cells, coinciding with increased Il22 transcription. (F) Flow cytometric analyses of RORγt expression in CRISPR/Cas9-edited primary human T cells differentiated in vitro using TH17 conditions. (G) qPCR analyses of expression of indicated genes in paired isogenic human TH17 cells that were engineered with CRISPR/Cas9 and either A20 ZF7 targeted or non-targeting guide RNAs. Note increased expression of TNFAIP3 , IL17A , and IL22 in A20 ZF7 ablated TH17 cells. Three pairs of isogenic samples from two healthy donors are shown. Data shown as mean + SEM. Statistics calculated using unpaired two-tailed Student t-test with Welch correction (B, C, E) or paired ratio t-test (G). *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, CRISPR, Two Tailed Test