Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Characterization of α-sarcoglycan WT and R77C fusion constructs and effect of bortezomib treatment. ( A ) Schematic representation of the cellular models showing that immortalized fibroblasts from a LGMD2D patient carrying the R77C homozygous mutation were transduced with lentivirus expressing WT-α-SGmCh or R77C-α-SGmCh constructs. ( B ) Confocal images of mCherry signal (red) and α-SG (green) detected by immunofluorescence in fibroblasts transduced with the lentivirus expressing WT-α-SGmCh under permeabilized (P) and non-permeabilized condition (NP). ( C ) Confocal images of mCherry signal (red) and α-SG (green) detected by immunofluorescence in fibroblasts transduced with the lentivirus expressing R77C-α-SGmCh under permeabilized (P) and non-permeabilized condition (NP) and following bortezomib (BTZ) treatment at 30 nM. Nuclei are labelled by Hoechst staining (blue). Scale bar = 20 µm.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Construct, Mutagenesis, Transduction, Expressing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: High-content screening for R77C-α-SGmCh membrane rescue. ( A ) Workflow for the high-content screening of R77C-α-SGmCh membrane rescue expression in 384 well plates. CPD = tested compounds. ( B ) mCherry fluorescent signal (red) and α-SG staining (green) in non permeabilized condition in fibroblasts overexpressing R77C-α-SGmCh treated with 0.1% DMSO and 30 nM bortezomib. Nuclei are labelled by Hoechst staining (blue). Scale bar = 50 µm. ( C , D ) Quantification of mCherry and membrane α-SG positive fibroblasts ( C ) and cell viability ( D ) following treatment with increasing concentrations of three proteasome inhibitors; bortezomib (black), carfilzomib (green) and MG132 (red). Values are expressed as percentage of the maximal response induced by bortezomib ( C ) or as percentage of the response induced by 0.1% DMSO ( D ) and each point represents the mean ± SD of four replicates. ( E ) High-throughput screening validation for the R77C-α-SGmCh membrane rescue in fibroblasts treated with the negative control, 0.1% DMSO, and the positive control, 30 nM bortezomib, in each of the 384-well plates of the screening. ( F ) Determination of the Z′ factor for each in each of the 384-well plates of the screening. ( G ) Primary screen cell-based assay for R77C-α-SGmCh membrane rescue. Dot plot representation of the effects of the 2560 drugs on R77C-α-SGmCh membrane expression (Z score \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ > $$\end{document} > 3) and cell viability (Viability \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ > $$\end{document} > 45%).; thiostrepton = THSP; bortezomib = BTZ, carfilzomib = CFZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: High Content Screening, Membrane, Expressing, Staining, High Throughput Screening Assay, Biomarker Discovery, Negative Control, Positive Control, Cell Based Assay

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Effect of the five identified compounds on membrane localization of R77C α-SG protein. ( A ) mCherry fluorescent signal (in red) and α-SG staining (in green) in non permeabilized condition in fibroblasts overexpressing R77C-α-SGmCh treated with 0.1% DMSO, bortezomib 30 nM and the five drugs identified at the dose of 5 µM (iodoacetamide, brefeldin A, BNTX, BBMP and thiostrepton). Nuclei are labelled by Hoechst staining (blue). Scale bar = 50 µm. ( B ) Quantification of mCherry and membrane α-SG positive cells and cell viability following treatment with increasing concentrations of bortezomib, iodoacetamide, brefeldin A, BNTX, BBMP and thiostrepton. Values are expressed as the percentage of the maximal response induced by bortezomib and each point represents the mean ± SD of four replicates; thiostrepton = THSP; bortezomib = BTZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Membrane, Staining

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Mechanism of action of Thiostrepton. ( A ) Measure of SGCA gene expression by qPCR in fibroblasts overexpressing R77C-α-SGmCh and treated with 0.1% DMSO, 3 µM thiostrepton, 30 nM bortezomib or 1 µM MG132 for 24 hours. Data are normalized to non-transduced fibroblasts. ( B ) Grouping of western blots analysis of α-SG and α-actin expression in non-treated (NT) fibroblasts overexpressing WT- and R77C-α-SGmCh or treated with thiostrepton (THSP, 3 µM) or bortezomib (BTZ, 30 nM) for 24 hours. Full-length blots are presented in Supplementary Fig. . ( C ) Quantification of trypsin-like, chymotrypsin-like and caspase-like activities of the proteasome in fibroblasts overexpressing R77C-α-SGmCh treated for 24 h with 0.1% DMSO, 3 µM thiostrepton, 30 nM bortezomib or 1 µM MG132.Values are expressed as the percentage of the response relative to 0.1% DMSO and each point represents the mean ± SD of four replicates.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Gene Expression, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Transcriptomic analysis. ( A ) The PCA graph of global gene expression data computed using Partek Genomic Suite and Partek Flow. 0.1% DMSO samples are shown as blue spheres; 3 µM thiostrepton samples in purple; 30 nM bortezomib samples in red and 1 µM MG132 samples in green. ( B ) Hierarchical clustering and heat map of significantly expressed genes in thiostrepton 3 µM vs 0.1% DMSO using RNA sequencing in fibroblasts overexpressing R77C-α-SGmCh. Color coding from green to red depicts gene expression differences between treatments from low to high expression, respectively. ( C ) Venn diagram illustrating the number of shared genes modulated in fibroblasts overexpressing R77C-α-SGmCh after 24 h of treatment with 0.1% DMSO, 3 µM thiostrepton, 30 nM bortezomib or 1 µM MG132. ( D ) Functional enrichment analysis of up-regulated genes in fibroblasts overexpressing R77C-α-SGmCh after 24 h of treatment with 30 nM bortezomib, 3 µM thiostrepton or 1 µM MG132 vs 0.1% DMSO. Analysis has been performed with the Panther pathway database. Values are expressed as combined scores and p-values are indicated; thiostrepton = THSP; bortezomib = BTZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Gene Expression, RNA Sequencing, Expressing, Functional Assay

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Synergistic effects of thiostrepton and bortezomib. Quantification of chymotrypsin-like activity of the proteasome ( A , B ) or mCherry and membrane α-SG expression ( C , D ) in fibroblasts overexpressing R77C-α-SGmCh. Cells were treated for 24 h with increasing concentration of thiostrepton in the presence of 3 nM, 5 nM or 15 nM bortezomib ( A , C ) or with increasing concentrations of bortezomib in the presence of 300 nM, 1 µM or 1.5 µM thiostrepton ( B , D ).Values are expressed as percentage of the response induced by 0.1% DMSO ( A , B ) or as percentage of the maximal response induced by bortezomib ( C , D ) and each point represents the mean ± SD of four replicates. ( E ) EC 50 of thiostrepton, bortezomib and combination treatments on membrane α-SGmCh membrane rescue. ( F ) Quantification of mCherry and membrane α-SG positive cells after 0.1% DMSO, 1.5 µM thiostrepton, 3 nM bortezomib and combination treatments. ***p ≤ 0.001; thiostrepton = THSP; bortezomib = BTZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Activity Assay, Membrane, Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Thiostrepton effect on the rescue of R77C-α-SGmCh protein in iPSC-derived myoblasts. ( A ) Differentiation of iPSCs into myoblasts following a 3-step protocol of differentiation. iPSC-derived myoblasts were transduced with the lentivirus overexpressing R77C-α-SGmCh and treated for 24 h with increasing concentrations of thiostrepton or negative and positive controls (0.1% DMSO and 30 nM bortezomib, respectively). ( B ) Quantification of the chymotrypsin-like activity of the proteasome following thiostrepton 3 µM treatment. Values are expressed as the percentage of the response induced by DMSO (0.1%) ( C ) Quantification of mCherry and membrane α-SG positive cells after thiostrepton 3 µM treatment. Values are expressed as percentage of the maximal response induced by bortezomib. ( D ) Images of mCherry fluorescent signal (red), membrane α-SG (green) and Hoechst staining (blue) in iPSC-derived myoblasts treated with 0.1% DMSO or thiostrepton 3 µM in non-permeabilized condition. Outsets represent higher magnification images. Scale bar = 50 µm; thiostrepton = THSP; bortezomib = BTZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Derivative Assay, Transduction, Activity Assay, Membrane, Staining

Journal: Scientific Reports

Article Title: Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

doi: 10.1038/s41598-019-43399-w

Figure Lengend Snippet: Evaluation of thiostrepton treatment on other missense α-SG mutants. Immortalized fibroblasts were transduced with lentivirus expressing WT-, R34H-, I124T- or V247M-α-SGmCh constructs and treated for 24 h with 0.1% DMSO or increasing concentrations of thiostrepton. The membrane α-SG expression was monitored by IF under non-permeabilized condition. ( A ) Confocal images of mCherry fluorescent signal (red), membrane α-SG (green) and Hoechst staining (blue) after 0.1% DMSO or THSP 3 µM treatments. Scale bar is 20 µm. ( B ) Quantification of mCherry and membrane α-SG positive cells following treatment with increasing concentrations of Thiostrepton. Values are expressed as percentage of mCherry and α-SG positive cells; thiostrepton = THSP; bortezomib = BTZ.

Article Snippet: Twenty-four hours after seeding, cells were treated with 30 nM of bortezomib (Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR).

Techniques: Transduction, Expressing, Construct, Membrane, Staining

Journal: Nature methods

Article Title: Single-cell western blotting

doi: 10.1038/nmeth.2992

Figure Lengend Snippet: Single-cell western blotting. ( a ) The scWestern array consists of thousands of microwells (20 µm diameter, 30 µm deep) patterned in a 30 µm-thick photoactive polyacrylamide gel seated on a glass microscope slide. The array is comprised of 16 blocks of 14×30 microwells (6,720 in total) cast against an SU-8 photoresist master fabricated by soft lithography. E: electric field. Scale bar: 10 mm. ( b ) Widefield micrograph of a microwell block containing 15 µm fluorescent microspheres (scale bar: 2 mm), and confocal micrograph of a live EGFP-expressing neural stem cell (NSC) settled in a rhodamine-tagged gel (GEL, scale bar: 10 µm). ( c ) Open-gel scWestern analysis is a 4 hour, 6 stage assay comprised of: cell settling, chemical lysis with a denaturing RIPA buffer, polyacrylamide gel electrophoresis (PAGE), UV-initiated protein immobilization onto the gel (hν: photon energy), diffusion-driven antibody probing (i.e., primary and fluorescently-labeled secondary antibody probes; 1° Ab and 2° Ab*), and fluorescence imaging. ( d ) PAGE resolves 5 fluorescently labeled proteins in a 550 µm separation distance (DRO, dronpa 27 kDa; OVA, ovalbumin 45 kDa; BSA, bovine serum albumin 66 kDa; OVA´, OVA dimer 90 kDa; BSA´, BSA dimer 132 kDa). ( e ) scWestern analysis of EGFP and β-tubulin (βTUB) from a single NSC (RFU: relative fluorescence units). Distinct fluorescent dyes on each secondary antibody enable multiplexed target analysis (EGFP: Alexa Fluor 488-labeled secondary antibody, βTUB: Alexa Fluor 555−). Chemical stripping and re-probing allows multiplexed scWestern analysis. Antibody details for all figures are in Online Methods .

Article Snippet: Cell cultures were imaged using a Nikon Eclipse T i inverted fluorescence microscope (Nikon Instruments) or an ImageXpress Micro XL Widefield High Content Screening System (Molecular Devices).

Techniques: Single Cell Western, Microscopy, Blocking Assay, Expressing, Lysis, Polyacrylamide Gel Electrophoresis, Diffusion-based Assay, Labeling, Fluorescence, Imaging, Stripping Membranes

Journal: Nature methods

Article Title: Single-cell western blotting

doi: 10.1038/nmeth.2992

Figure Lengend Snippet: Single-cell western blotting. ( a ) The scWestern array consists of thousands of microwells (20 µm diameter, 30 µm deep) patterned in a 30 µm-thick photoactive polyacrylamide gel seated on a glass microscope slide. The array is comprised of 16 blocks of 14×30 microwells (6,720 in total) cast against an SU-8 photoresist master fabricated by soft lithography. E: electric field. Scale bar: 10 mm. ( b ) Widefield micrograph of a microwell block containing 15 µm fluorescent microspheres (scale bar: 2 mm), and confocal micrograph of a live EGFP-expressing neural stem cell (NSC) settled in a rhodamine-tagged gel (GEL, scale bar: 10 µm). ( c ) Open-gel scWestern analysis is a 4 hour, 6 stage assay comprised of: cell settling, chemical lysis with a denaturing RIPA buffer, polyacrylamide gel electrophoresis (PAGE), UV-initiated protein immobilization onto the gel (hν: photon energy), diffusion-driven antibody probing (i.e., primary and fluorescently-labeled secondary antibody probes; 1° Ab and 2° Ab*), and fluorescence imaging. ( d ) PAGE resolves 5 fluorescently labeled proteins in a 550 µm separation distance (DRO, dronpa 27 kDa; OVA, ovalbumin 45 kDa; BSA, bovine serum albumin 66 kDa; OVA´, OVA dimer 90 kDa; BSA´, BSA dimer 132 kDa). ( e ) scWestern analysis of EGFP and β-tubulin (βTUB) from a single NSC (RFU: relative fluorescence units). Distinct fluorescent dyes on each secondary antibody enable multiplexed target analysis (EGFP: Alexa Fluor 488-labeled secondary antibody, βTUB: Alexa Fluor 555−). Chemical stripping and re-probing allows multiplexed scWestern analysis. Antibody details for all figures are in Online Methods .

Article Snippet: Cell cultures were imaged using a Nikon Eclipse T i inverted fluorescence microscope (Nikon Instruments) or an ImageXpress Micro XL Widefield High Content Screening System (Molecular Devices).

Techniques: Single Cell Western, Microscopy, Blocking Assay, Expressing, Lysis, Polyacrylamide Gel Electrophoresis, Diffusion-based Assay, Labeling, Fluorescence, Imaging, Stripping Membranes

Journal: mBio

Article Title: Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection

doi: 10.1128/mBio.02342-21

Figure Lengend Snippet: Knockout of ZDHHC17 decreases the SADS-CoV replication. (A) The target sequence in clonal cells is amplified and cloned into the pGEM-T-EASY vector. (Upper) Edited nucleotide sequences in the ZD17 gene alleles are shown according to sequencing analysis. (Lower) The clonal HeLa-ZD17 KO and HeLa cells were cultured in 6-well plates, and the expression of endogenous ZD17 was detected by Western blotting with anti-ZD17 rabbit polyclonal antibody. (B) HeLa and HeLa-ZD17 KO cells were cultured in 24-well plates and infected with SADS-CoV (MOI, 0.1). At different time points (2, 12, 24, 48, and 72 hpi), RNA was extracted from supernatants and viral genome copies were determined by RT-qPCR with primers targeting the SADS-CoV RdRp gene. (C) Cells from panel B were fixed at 12, 24, and 48 hpi, respectively, and analyzed by IFA using an anti-N protein antibody. (D) The infection rates in panel C were quantified with high content analysis. (E) At 24 h and 48 hpi, CPE was examined to compare the production of infectious progeny virus. (F) The real-time growth and adhesion kinetics of HeLa and HeLa-ZD17 KO cells were monitored using a label-free cell-based assay by the xCELLigence real-time cellular analysis (RTCA) system.

Article Snippet: The following antibodies were used in this study: ZDHHC17 polyclonal antibody (15465-1-AP; Proteintech), DYKDDDK-Tag(3B9) mouse antibody (M20008; Abmart), anti-dsRNA MAb J2 (J2-1702; Scicons, Hungary), Cy3-conjugated anti-rabbit IgG (ab6939; Abcam), and DyLight488 anti-mouse IgG (ab96879; Abcam).

Techniques: Knock-Out, Sequencing, Amplification, Clone Assay, Plasmid Preparation, Cell Culture, Expressing, Western Blot, Infection, Quantitative RT-PCR, High Content Screening, Virus, Cell Based Assay

Journal: mBio

Article Title: Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection

doi: 10.1128/mBio.02342-21

Figure Lengend Snippet: ZDHHC17 is involved in viral RNA synthesis. HeLa and HeLa-ZD17 KO cells were inoculated with SADS-CoV (MOI, 0.1) at 4°C for 1 h and washed with cold PBS. (A) The cells were harvested and viral RNA was extracted for determining the virion attachment at the cell surface. (B) The infected cells as described above were further cultured at 37°C for another 1 h. Cells were then harvested after pronase treatment and viral RNA was extracted for determining the virion internalization. (C) At 24 hpi, the ratio of SADS-CoV RNA copy number in the supernatants versus the cell lysates were separately determined by RT-qPCR for assembly and release assay. (D) Quantification of extracellular genomic RNA. (E) Quantification of intracellular positive- and negative-strand RNA at 0, 2, 4, 6, 8, 10, and 12 hpi. (F) Viral RNA was assessed by staining cells with anti-dsRNA antibody followed by confocal microscopy analysis. (G) The infectious virions secreted from HeLa and HeLa-ZD17 KO cells were determined by 50% tissue culture infectious dose assays in Vero cells.

Article Snippet: The following antibodies were used in this study: ZDHHC17 polyclonal antibody (15465-1-AP; Proteintech), DYKDDDK-Tag(3B9) mouse antibody (M20008; Abmart), anti-dsRNA MAb J2 (J2-1702; Scicons, Hungary), Cy3-conjugated anti-rabbit IgG (ab6939; Abcam), and DyLight488 anti-mouse IgG (ab96879; Abcam).

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Release Assay, Staining, Confocal Microscopy

Journal: Saudi Journal of Biological Sciences

Article Title: Acaudina molpadioides mediates lipid uptake by suppressing PCSK9 transcription and increasing LDL receptor in human liver cells

doi: 10.1016/j.sjbs.2021.08.003

Figure Lengend Snippet: The correlation of PCSK9 and statin in regulating the metabolism of LDL-C in the PCSK9 and LDLR are synthesized in the liver. The secreted PCSK9 protein binds to the extracellular domain of LDLR at the cell surface. The PCSK9/LDLR-LDL-C complex enters via the endosomal pathway and is directed to the lysosomal compartment for degradation of both PCSK9 and LDLR, decreasing the number of LDLRs available for clearance of LDL-C particles (A). The gene expression of PCSK9 is subjected to the regulation by the binding of SREBP2, HNF1 and PPAR to their corresponding binding sites on PCSK9 promoter (B). Activation of SREBP2, under conditions of intracellular cholesterol depletion due to inhibitory activity of statin, increases the expression of both PCSK9 and LDLR (C).

Article Snippet: The uptake of LDL-C by LDLR was determined by using LDL uptake assay kit (Abcam®) and was carried out according to the manufacturer’s instructions.

Techniques: Synthesized, Expressing, Binding Assay, Activation Assay, Activity Assay

Journal: Saudi Journal of Biological Sciences

Article Title: Acaudina molpadioides mediates lipid uptake by suppressing PCSK9 transcription and increasing LDL receptor in human liver cells

doi: 10.1016/j.sjbs.2021.08.003

Figure Lengend Snippet: LDL-C uptake and LDLR protein expression on HepG2 cells treated with 25 µg/mL A. molpadioides EFA and EFB, 20 µM berberine sulphate as positive control and 1% (v/v) dimethyl sulfoxide (DMSO) as negative control. HepG2 cells were treated with the mediators for 24 h and the LDL-C uptake was observed. At the end of the treatment duration, the culture medium was replaced with 100 µL/well LDL-DyLight™ 550 working solution and incubated for additional 3 to 4 h. Stained cells were observed with high content screening (HCS) with filters capable of measuring excitation and emission wavelengths 540 and 570 nm, and acquired with MetaXpress® 5.1. EFA treatment produced the highest intensity in cells stained with LDL-DyLight™ 550 (yellow fluorescence) indicating an increase in LDL-C uptake as compared to EFB treatment, in line with the increase in LDLR expression (green fluorescence). For LDLR expression, HepG2 cells were incubated for one hour with 100 µL/well of diluted Rabbit Anti-LDL Receptor Primary Antibody and were subsequently incubated in the dark for one hour with 100 µL/well of diluted DyLight™ 488-Conjugated Secondary Antibody. Stained cells were observed with high content screening (HCS) fluorescein detection (excitation/emission = 485/535 nm) and acquired with MetaXpress® 5.1. Fluorescence intensity was analysed and measured with ImageJ. Scale bar: 50 µm.

Article Snippet: The uptake of LDL-C by LDLR was determined by using LDL uptake assay kit (Abcam®) and was carried out according to the manufacturer’s instructions.

Techniques: Expressing, Positive Control, Negative Control, Incubation, Staining, High Content Screening, Produced, Fluorescence

Journal: Saudi Journal of Biological Sciences

Article Title: Acaudina molpadioides mediates lipid uptake by suppressing PCSK9 transcription and increasing LDL receptor in human liver cells

doi: 10.1016/j.sjbs.2021.08.003

Figure Lengend Snippet: The effect of A. molpadioides enhanced fractions A and B (EFA and EFB) on LDL-C uptake (A) and LDLR translocation (B). ImageJ analysis was carried out by measuring the fold change value of the fluorescence intensity of treated cells over the fluorescence intensity of the untreated cells or negative control, 1% (v/v) dimethyl sulfoxide (DMSO). EFA treatment induced the highest level of LDL-C uptake followed by the positive control, 20 µM berberine sulphate (BBR). Collectively, EFA appeared to show better potential in upregulating LDL-C uptake concomitantly with an increase in LDLR as compared to EFB. Multiple groups were analysed with one-way analysis of variance (ANOVA) where means with different letters were separated with Duncan’s multiple range test group at p < 0.05.

Article Snippet: The uptake of LDL-C by LDLR was determined by using LDL uptake assay kit (Abcam®) and was carried out according to the manufacturer’s instructions.

Techniques: Translocation Assay, Fluorescence, Negative Control, Positive Control