Journal: Endocrine-Related Cancer

Article Title: Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells

doi: 10.1530/ERC-23-0010

Figure Lengend Snippet: SEMA3C upregulates lipogenic and cholesterogenic enzymes. (A) LNCaP cells treated with recombinant SEMA3C 1 μM in 5% CSS-supplemented phenol red-free RPMI for 24 h. mRNA expression of genes involved in cholesterol pathway by RT-qPCR method. SEMA3C was silenced in (B) C4-2 cells and (C) 22Rv1 cells by siRNA and mRNA expression of cholesterogenic genes was quantified in siSEMA3C-treated cells compared to respective control cells. (D) Increased activity of SREBP-responsive element in transiently transfected luciferase reporter plasmid into LNCaP cells followed by recombinant SEMA3C 1 μM or vehicle treatment ( n = 6). (E) LNCaP cells were treated with erlotinib 2 μM approximately 2 h prior to the treatment with recombinant SEMA3C 1 μM. After 24 h, SREBP-responsive promoter activity was assessed by luciferase assay. (F) qPCR was performed to quantify relative mRNA expression of a panel of SREBP targets in LNCaP cells in the presence of erlotinib 2 μM. Only HMGCS1 mRNA expression level was affected. (G) The whole protein of LNCaP SEMA3C and LNCaP vector was immunoblotted to examine the protein level of genes involved in the cholesterol pathway. (mean ± s.d. ; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Primary antibodies include AR (Santa Cruz Biotechnology, Cat. No. sc-816), PSA (Cell Signaling Technology, Cat. No. 5877), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), AKR1C3 (Abcam, Cat. No. ab49680), HSD3B2 (Abnova, Taipei, Taiwan; Cat. No. 3284-mo2), HSD17B3 (Abnova, Cat. No. 29-119), FASN (Cell Signaling Technology, Cat. No. 3180), HMGCS1 (Cell Signaling Technology, Cat. No. 36877), SREBP-1 (Santa Cruz Biotechnology, Cat. No. sc-13551), UGT2B17 (Abcam, Cat. No. ab126269), and actin (Sigma-Aldrich, Cat. No. A2066).

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Control, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Cell death & disease

Article Title: High glucose levels promote glycolysis and cholesterol synthesis via ERRα and suppress the autophagy-lysosomal pathway in endometrial cancer.

doi: 10.1038/s41419-025-07499-y

Figure Lengend Snippet: Fig. 3 After stimulation by high glucose, the role of ERRα in regulating glucose and cholesterol metabolism. A Heatmap of the average read density and chromatin occupancy of the transcription start site (TSS) of ERRα in two independent samples of HEC-1A cells with control or upregulated ERRα. B, C Distribution of CUT&Tag peaks of ERRα in the genomic region from the second repetition of control samples. D, E Distribution of ERRα sites relative to HK2 and HMGCS1 promoters based on the CUT&Tag analysis. F HEC-1A and KLE EC cells transduced with si-ERRα were treated with NG or HG for 48 h. The expression of ERRα, HK2, PKM2, HMGCR, and HMGCS1 was measured by western blotting (WB), with β-actin used as a loading control. G HEC-1A and KLE EC cells with ov-ERRα were subjected to NG or HG for 48 h. The expression of ERRα, HK2, PKM2, HMGCR, and HMGCS1 was measured by western blotting, with β-actin used as a loading control. H, I Analysis of the data in panel F comparing the differences in protein expression among the si-ERRα-NG, con-HG, and si-ERRα-HG groups using the con- NG group as the reference baseline. J, K Analysis of the data in panel G comparing the differences in protein expression among the con-NG, ov-ERRα-HG, and con-HG groups using the ov-ERRα-NG group as the reference baseline. L–O HEC-1A and KLE EC cells were treated with Mnt, NG, or HG for 48 h, after which the lactate or TC concentrations were measured with three replications, respectively. P, S HEC-1A and KLE EC cells with or without ERRα knockdown were treated with NG or HG for 48 h, after which the lactate or TC concentration was measured with three replications, respectively. *P < 0.05, **P < 0.01 or ***P < 0.001. NG normal glucose, HG high glucose, EC endometrial cancer, TC total cholesterol.

Article Snippet: Membranes were incubated with antibodies specific for ERRα (1:500, #ab137489, Abcam, MA, USA), HK2 (1:1000, #66974- 1-Ig, Proteintech, Wuhan, China), HK2, (#ab209847, Abcam, MA, USA), PKM2 (1:1000, #60268-1-Ig, Proteintech, Wuhan, China), HMGCS1 (1:1000, #17643-1- AP, Proteintech, Wuhan, China), HMGCR (1:1000, #A16875, ABclonal, Wuhan, China), p62 (1:1000, #ab207305, Abcam, MA, USA) and β-actin (1:1000, #YM8343, ImmunoWay, Suzhou, China); washed; and incubated with a goat anti-rabbit secondary antibody (1:10,000, #SA00001-2, Proteintech, Wuhan, China) or goat anti-mouse secondary antibody (1:10,000, #SA00001-1, Proteintech, Wuhan, China) for 2 h. The membranes were washed 30min.

Techniques: Control, Transduction, Expressing, Western Blot, Knockdown, Concentration Assay

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. Venn diagram representing the significant overexpressed genes (> 1.5 fold-change) in the 16- hours CSC-enriched cultures compared to individual adherent monolayer cultures for each cell line. Overall analysis of the RNA-sequencing data for MCF-7, T47D and MDA-MB-231 cell lines, identified 344, 243 and 477 genes. B. Representation of the pathway-associated networks significantly above the statistical threshold (p<0.001) in the CSC-enriched cell line subpopulations. C. Schematic diagram of several mevalonate pathway enzymatic intermediates. D. Single-cell expression analysis of mevalonate pathway genes by qPCR, conducted in MCF-7 (blue, n = 76), T47D (green, n = 88) and MDA-MB-231 (MDA-231) (red, n = 83) adherent monolayer cultures. Mean gene expression ±SEM per single cell for each cell line is represented on left y-axis and the percentage of cells expressing a particular gene within the overall single-cell population is represented on the right y-axis. E. Percentage of cells expressing HMGCS1 in adherent monolayer and CSC-enriched (CSC-en) single cell populations, in MCF-7 (blue), T47D (green) and MDA-MB-231 (red) cell lines. F. Line graphs depicting single-cell gene associations using Spearman’s correlation to identify significant correlations between mevalonate pathway genes in each cell line monolayer population (p<0.05, ρ≥0.4). Each significant gene correlation is represented by a line and connecting lines are weighted in an increasing manner to represent increasing correlation coefficient values . G. Relative gene expression of mevalonate pathway genes in the CSC subpopulations compared to monolayer controls from the original RNA-sequencing data.

Article Snippet: Briefly, deparaffinized sections were subjected to antigen retrieval by high pressure cooking and DIVA antigen retrieval, followed by blocking with hydrogen peroxide and incubation with primary antibody against HMGCS1 (Atlas Antibodies, Stockholm, Sweden, 1:500).

Techniques: RNA Sequencing, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. HMGCS1 gene expression measured by qPCR in MCF-7, T47D and MDA-MB-231 (MDA-231) cell lines cultured as adherent monolayer or 16 hour CSC-enriched (CSC-en) cultures. HMGCS1 transcript levels are relative to individual monolayer controls. Mean ±SEM from three biological replicates are shown. B. HMGCS1 protein expression in MCF-7, T47D and MDA-MB-231 cell lines cultured as adherent monolayers, 16 hours suspension cultures or 5-days mammosphere cultures. A representative western blot and associated densitometry are shown. C. Representative western blot and associated densitometry of MCF-7, T47D and MDA-MB-231 cell lines transfected with HMGCS1 siRNA confirming the knockdown of HMGCS1 protein expression, relative to the scrambled negative control. β-actin was used as a loading control. D. Percentage of viable cells in 16 hours CSC-enriched cultures of MCF-7, T47D and MDA-MB-231 cells transfected with either scrambled negative control or HMGCS1 siRNA. E. Mammosphere forming capacity (MFC) of the three cell lines transfected with either scrambled negative control or HMGCS1 siRNA, represented as percentage of total cells seeded. F. Representative western blot and associated densitometry confirming the increase of HMGCS1 protein in cells treated 48 hours with 1 μM Simvastatin (SS), compared to the control (DMSO) samples for each cell line. G. Percentage of viable cells in 16 hours CSC-enriched cultures of MCF-7, T47D and MDA-MB-231 cells treated with either DMSO control or 1 μM Simvastatin. H. Mammosphere forming capacity (MFC) of the three cell lines treated with either DMSO control or 1 μM Simvastatin. In all graphs the mean ±SEM of three independent experiments is shown and, the significance level was determined by two-tailed, paired Student’s t-test; *p<0.05, **p<0.01.

Article Snippet: Briefly, deparaffinized sections were subjected to antigen retrieval by high pressure cooking and DIVA antigen retrieval, followed by blocking with hydrogen peroxide and incubation with primary antibody against HMGCS1 (Atlas Antibodies, Stockholm, Sweden, 1:500).

Techniques: Gene Expression, Cell Culture, Expressing, Suspension, Western Blot, Transfection, Knockdown, Negative Control, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. RT-qPCR analysis of the transcript levels of proliferation, pluripotency and EMT/stem-like related genes for MCF-7, T47D and MDA-MB-231 (MDA-231) cells transfected with either scrambled negative control or HMGCS1 siRNA. Expression levels for each individual gene were normalized to the scrambled negative control for each cell line. Graphs show mean ±SEM, n = 3, *p<0.05, **p<0.01 by two-tailed paired Student’s t-test. B. Single-cell gene expression profiling in single cells expressing HMGCS1 (blue, green, red) versus cells which do not express HMGCS1 (black), of adherent monolayer cultures for MCF-7 (blue, n = 76), T47D (green, n = 88) and MDA-MB-231 (red, n = 83) cell lines. Mean ±SEM is shown. The Mann-Whitney U test was used to analyse expression of individual genes between two groups and obtained p-values were Bonferroni-adjusted to correct for multiple testing . C. Spearman’s correlation analysis was conducted on single-cell populations which did not express HMGCS1 (MCF-7, n = 54; T47D, n = 63; and MDA-MB-231, n = 62) or expressed HMGCS1 (MCF-7, n = 22; T47D, n = 25; and MDA-MB-231, n = 21), respectively. Correlations were drawn between the same proliferation, pluripotency and EMT/stem-like genes as analysed in A ( – Tables). Connecting lines were weighted in an increasing manner to represent increasing correlation coefficient values (p<0.05, ρ≥0.4).

Article Snippet: Briefly, deparaffinized sections were subjected to antigen retrieval by high pressure cooking and DIVA antigen retrieval, followed by blocking with hydrogen peroxide and incubation with primary antibody against HMGCS1 (Atlas Antibodies, Stockholm, Sweden, 1:500).

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Expressing, Two Tailed Test, Gene Expression, MANN-WHITNEY

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. HMGCS1 expression in a tissue microarray (TMA) from 149 patients with lymph-node positive breast cancer. B. Comparative pie charts for each HMGCS1 subgroup (negative, low, intermediate and high) showing the frequency of ERα-positive and ERα-negative tumour subtypes present in each. C. Percentage of p53 mutations in ERα-positive and ERα-negative breast cancers for HMGCS1 subgroups.

Article Snippet: Briefly, deparaffinized sections were subjected to antigen retrieval by high pressure cooking and DIVA antigen retrieval, followed by blocking with hydrogen peroxide and incubation with primary antibody against HMGCS1 (Atlas Antibodies, Stockholm, Sweden, 1:500).

Techniques: Expressing, Microarray

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: Clinical associations of HMGCS1 in a lymph node positive breast cancer patient cohort.

Article Snippet: Briefly, deparaffinized sections were subjected to antigen retrieval by high pressure cooking and DIVA antigen retrieval, followed by blocking with hydrogen peroxide and incubation with primary antibody against HMGCS1 (Atlas Antibodies, Stockholm, Sweden, 1:500).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. Venn diagram representing the significant overexpressed genes (> 1.5 fold-change) in the 16- hours CSC-enriched cultures compared to individual adherent monolayer cultures for each cell line. Overall analysis of the RNA-sequencing data for MCF-7, T47D and MDA-MB-231 cell lines, identified 344, 243 and 477 genes. B. Representation of the pathway-associated networks significantly above the statistical threshold (p<0.001) in the CSC-enriched cell line subpopulations. C. Schematic diagram of several mevalonate pathway enzymatic intermediates. D. Single-cell expression analysis of mevalonate pathway genes by qPCR, conducted in MCF-7 (blue, n = 76), T47D (green, n = 88) and MDA-MB-231 (MDA-231) (red, n = 83) adherent monolayer cultures. Mean gene expression ±SEM per single cell for each cell line is represented on left y-axis and the percentage of cells expressing a particular gene within the overall single-cell population is represented on the right y-axis. E. Percentage of cells expressing HMGCS1 in adherent monolayer and CSC-enriched (CSC-en) single cell populations, in MCF-7 (blue), T47D (green) and MDA-MB-231 (red) cell lines. F. Line graphs depicting single-cell gene associations using Spearman’s correlation to identify significant correlations between mevalonate pathway genes in each cell line monolayer population (p<0.05, ρ≥0.4). Each significant gene correlation is represented by a line and connecting lines are weighted in an increasing manner to represent increasing correlation coefficient values . G. Relative gene expression of mevalonate pathway genes in the CSC subpopulations compared to monolayer controls from the original RNA-sequencing data.

Article Snippet: HMGCS1 gene expression was silenced with 30 nM HMGCS1 siRNA or treated with Trilencer-27 Universal Scrambled Negative Control siRNA (Origene Technologies Inc., Rockville, MD, USA) for 72 hours using the Viromer Blue transfection system (Lipocalyx, Halle, Germany).

Techniques: RNA Sequencing, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. HMGCS1 gene expression measured by qPCR in MCF-7, T47D and MDA-MB-231 (MDA-231) cell lines cultured as adherent monolayer or 16 hour CSC-enriched (CSC-en) cultures. HMGCS1 transcript levels are relative to individual monolayer controls. Mean ±SEM from three biological replicates are shown. B. HMGCS1 protein expression in MCF-7, T47D and MDA-MB-231 cell lines cultured as adherent monolayers, 16 hours suspension cultures or 5-days mammosphere cultures. A representative western blot and associated densitometry are shown. C. Representative western blot and associated densitometry of MCF-7, T47D and MDA-MB-231 cell lines transfected with HMGCS1 siRNA confirming the knockdown of HMGCS1 protein expression, relative to the scrambled negative control. β-actin was used as a loading control. D. Percentage of viable cells in 16 hours CSC-enriched cultures of MCF-7, T47D and MDA-MB-231 cells transfected with either scrambled negative control or HMGCS1 siRNA. E. Mammosphere forming capacity (MFC) of the three cell lines transfected with either scrambled negative control or HMGCS1 siRNA, represented as percentage of total cells seeded. F. Representative western blot and associated densitometry confirming the increase of HMGCS1 protein in cells treated 48 hours with 1 μM Simvastatin (SS), compared to the control (DMSO) samples for each cell line. G. Percentage of viable cells in 16 hours CSC-enriched cultures of MCF-7, T47D and MDA-MB-231 cells treated with either DMSO control or 1 μM Simvastatin. H. Mammosphere forming capacity (MFC) of the three cell lines treated with either DMSO control or 1 μM Simvastatin. In all graphs the mean ±SEM of three independent experiments is shown and, the significance level was determined by two-tailed, paired Student’s t-test; *p<0.05, **p<0.01.

Article Snippet: HMGCS1 gene expression was silenced with 30 nM HMGCS1 siRNA or treated with Trilencer-27 Universal Scrambled Negative Control siRNA (Origene Technologies Inc., Rockville, MD, USA) for 72 hours using the Viromer Blue transfection system (Lipocalyx, Halle, Germany).

Techniques: Gene Expression, Cell Culture, Expressing, Suspension, Western Blot, Transfection, Knockdown, Negative Control, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. RT-qPCR analysis of the transcript levels of proliferation, pluripotency and EMT/stem-like related genes for MCF-7, T47D and MDA-MB-231 (MDA-231) cells transfected with either scrambled negative control or HMGCS1 siRNA. Expression levels for each individual gene were normalized to the scrambled negative control for each cell line. Graphs show mean ±SEM, n = 3, *p<0.05, **p<0.01 by two-tailed paired Student’s t-test. B. Single-cell gene expression profiling in single cells expressing HMGCS1 (blue, green, red) versus cells which do not express HMGCS1 (black), of adherent monolayer cultures for MCF-7 (blue, n = 76), T47D (green, n = 88) and MDA-MB-231 (red, n = 83) cell lines. Mean ±SEM is shown. The Mann-Whitney U test was used to analyse expression of individual genes between two groups and obtained p-values were Bonferroni-adjusted to correct for multiple testing . C. Spearman’s correlation analysis was conducted on single-cell populations which did not express HMGCS1 (MCF-7, n = 54; T47D, n = 63; and MDA-MB-231, n = 62) or expressed HMGCS1 (MCF-7, n = 22; T47D, n = 25; and MDA-MB-231, n = 21), respectively. Correlations were drawn between the same proliferation, pluripotency and EMT/stem-like genes as analysed in A ( – Tables). Connecting lines were weighted in an increasing manner to represent increasing correlation coefficient values (p<0.05, ρ≥0.4).

Article Snippet: HMGCS1 gene expression was silenced with 30 nM HMGCS1 siRNA or treated with Trilencer-27 Universal Scrambled Negative Control siRNA (Origene Technologies Inc., Rockville, MD, USA) for 72 hours using the Viromer Blue transfection system (Lipocalyx, Halle, Germany).

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Expressing, Two Tailed Test, Gene Expression, MANN-WHITNEY

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: A. HMGCS1 expression in a tissue microarray (TMA) from 149 patients with lymph-node positive breast cancer. B. Comparative pie charts for each HMGCS1 subgroup (negative, low, intermediate and high) showing the frequency of ERα-positive and ERα-negative tumour subtypes present in each. C. Percentage of p53 mutations in ERα-positive and ERα-negative breast cancers for HMGCS1 subgroups.

Article Snippet: HMGCS1 gene expression was silenced with 30 nM HMGCS1 siRNA or treated with Trilencer-27 Universal Scrambled Negative Control siRNA (Origene Technologies Inc., Rockville, MD, USA) for 72 hours using the Viromer Blue transfection system (Lipocalyx, Halle, Germany).

Techniques: Expressing, Microarray

Journal: PLoS ONE

Article Title: The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer

doi: 10.1371/journal.pone.0236187

Figure Lengend Snippet: Clinical associations of HMGCS1 in a lymph node positive breast cancer patient cohort.

Article Snippet: HMGCS1 gene expression was silenced with 30 nM HMGCS1 siRNA or treated with Trilencer-27 Universal Scrambled Negative Control siRNA (Origene Technologies Inc., Rockville, MD, USA) for 72 hours using the Viromer Blue transfection system (Lipocalyx, Halle, Germany).

Techniques: Mutagenesis

Journal: PLoS Biology

Article Title: Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis

doi: 10.1371/journal.pbio.1000598

Figure Lengend Snippet: (A) The Sterol biosynthesis pathway shown in KEGG notation with abbreviated metabolites (abbreviations listed in ). The geranylgeranylation pathway responsible for GGPP synthesis is shown in the dashed box. (B) Heat map of the cholesterol biosynthesis temporal genes' expression during the first 12 h of mCMV infection (left panel) or IFNγ treatment (right panel). Each time point corresponds to one independent biological sample, and columns indicate time in hours. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.8× lower expression (dark blue) to a 1.2× higher expression (bright yellow). (C–H) Expression analysis measured by qRT-PCR of Hmgcs1 , Hmgcr , Idi1 , and Sqle genes in BMDM infected with mCMV(24 hpi) (C) or treated for 24 h with IFNγ (10 and 100 U/ml) (D), IFNβ (10 and 25 U/ml) (E), IL6 (10 and 25 U/ml) (F), IL1β (10 and 100 U/ml) (G), or TNF (10 and 100 U/ml) (H). Graphs show levels of mRNA expression of the respective genes either infected or cytokines-treated relative to mock samples. Bars represent the means ± SD of five independent experiments with biological triplicates for each experiment. * p <0.05, ** p <0.01, *** p <0.001, determined with an unpaired Student's t test.

Article Snippet: Taqman Primer probe sets were purchased from Applied Biosystems, Warrington, UK (Assay ID: Hmgcs1: Mm00524111-m1; Hmgcr: Mm01282499-m1; Idi1: Mm00836417-g1; Sqle: Mm00436772-A1).

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: PLoS Biology

Article Title: Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis

doi: 10.1371/journal.pbio.1000598

Figure Lengend Snippet: (A) Comparison by Western blot analysis of HMGCS1, HMGCR, and SQLE protein levels in mCMV infected (24 hpi) or mock-treated BMDM. Infection was measured by detection of the IE1 mCMV antigen. Intensity values relative to β-actin calculated by densitometry show a decrease of the total amount of protein in the mCMV-infected BMDM compared to the mock-treated samples of 64% for HMGCS1, 50% for HMGCR, and 85% for SQLE. Graphs are representative of two independent experiments with biological duplicates and triplicates, respectively. (B–C) Free cholesterol concentration was determined experimentally by enzymatic assay at 0, 6, 24, 48, and 72 hpi in BMDM (B) and NIH/3T3 cells (C). Cholesterol content is presented as the percentage of free intracellular cholesterol concentration from infected cells compared to mock treatment. Graphs represent the means ± SD of three independent experiments with biological quadruplicates for each experiment. (D) Free cholesterol concentration in BMDM cultures treated with varying concentrations of IFNγ, IFNβ, TNF, IL1β, or IL6. The cholesterol concentration was measured as mentioned above after 48 h post-cytokine treatment. Bars represent means ± SD of two independent experiments with biological quadruplicates for each experiment. * p <0.05, ** p <0.01, *** p <0.001, determined with an unpaired Student's t test.

Article Snippet: Taqman Primer probe sets were purchased from Applied Biosystems, Warrington, UK (Assay ID: Hmgcs1: Mm00524111-m1; Hmgcr: Mm01282499-m1; Idi1: Mm00836417-g1; Sqle: Mm00436772-A1).

Techniques: Comparison, Western Blot, Infection, Concentration Assay, Enzymatic Assay

Journal: PLoS Biology

Article Title: Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis

doi: 10.1371/journal.pbio.1000598

Figure Lengend Snippet: (A) NIH/3T3 cells were infected with mCMV-GFP (MOI of 0.2) and subsequently treated with Simvastatin (SMV) (2.5 µM) and mevalonate (MEV) (300 µM) or geranylgeraniol (GGOH) (15 µM) or farnesol (FOH) (15 µM) or squalene (SQE) (15 µM) or of water soluble cholesterol (complexes of cholesterol with methyl-β-cyclodextrin, CHO/MCD) (5 µg/ml) for 72 h. The level of infection was determined by measuring GFP fluorescence at 76 hpi . Graph represents the relative level of infection compared to the untreated cells, and bars represent mean values ± SD of three independent experiments with five biological replicates for each experiment. (B) NIH/3T3 cells were transfected for 48 h with either non-targeted, eGFP, Hmgcs1, Hmgcr, Sqle, Fdft1, or Dhcr7 On-target plus siRNA smart pool and then infected with mCMV-GFP (MOI of 0.2). (C) NIH/3T3 cells were transfected for 48 h with either Risc Free, M54, and M86 (knocking down mCMV viral genes), Hmgcr, Dhcr7, Fdps, Fntb, Pggt1b, or Rabggtb On-target plus siRNA smart pool, and then infected with mCMV-GFP (MOI of 0.2). The level of non-targeted siRNA (B) and Risc Free (C) treated cells was used as a baseline estimate for the cutoff point (two standard deviations and a p value <0.001 (determined with an unpaired Student's t test) as significant). Bars represent means ± SD of two independent experiments with three biological replicates for each experiment. (D) NIH/3T3 cells were incubated with various doses of IFNβ for 18 h in the presence or absence of 15 and 150 µM GGOH. The graph represents the inhibition of viral replication (in percentages) as a function of drug concentration. Bars represent mean ± SD of biological triplicates for each experiment. * p <0.05, ** p <0.01, *** p <0.001, determined with an unpaired Student's t test.

Article Snippet: Taqman Primer probe sets were purchased from Applied Biosystems, Warrington, UK (Assay ID: Hmgcs1: Mm00524111-m1; Hmgcr: Mm01282499-m1; Idi1: Mm00836417-g1; Sqle: Mm00436772-A1).

Techniques: Infection, Fluorescence, Transfection, Incubation, Inhibition, Concentration Assay

Journal: PLoS Biology

Article Title: Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis

doi: 10.1371/journal.pbio.1000598

Figure Lengend Snippet: (A) Heat map of expression levels of a set of genes after 24 h mock treatment, infection with Herpes simplex virus 1 (HSV1), Semliki forest virus (SFV), Vaccinia virus (VV), or Adenovirus (Ad) in BMDM . Genes represent the innate immunity activation, the MHC class II antigen presentation, and the cholesterol and unsaturated fatty acids biosynthesis. Each square represents a single biological replicate. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.4× lower expression (dark blue) to a 1.6× higher expression (bright yellow). (B) Expression analysis measured by qRT-PCR of Hmgcs1 , Hmgcr , Idi1 , and Sqle genes in BMDM after 24 h mock treatment, mCMV, or mCMVdie3 infection, respectively. Graphs show the level of expression of the indicated genes relative to mock-treated samples and bars represent mean ± SD of two independent experiments with triplicate biological measurements for each experiment. (C) BMDM were infected with mCMV or mock treated, and supernatant was collected after 8 h and directly added to fresh BMDM. After 24 h, RNA from these cultures was collected and Hmgcs1 , Hmgcr , and Sqle expression were measured by qRT-PCR. To test for the presence of any detectable virus, an aliquot of the supernatant was used to perform a standard plaque assay (no infectious virus detected, unpublished data). Graphs show the level of expression of the indicated genes relative to mock-treated samples and bars represent means ± SD of three independent experiments with triplicate biological measurements for each experiment. * p <0.05, ** p <0.01, *** p <0.001, determined with an unpaired Student's t test.

Article Snippet: Taqman Primer probe sets were purchased from Applied Biosystems, Warrington, UK (Assay ID: Hmgcs1: Mm00524111-m1; Hmgcr: Mm01282499-m1; Idi1: Mm00836417-g1; Sqle: Mm00436772-A1).

Techniques: Expressing, Infection, Virus, Activation Assay, Immunopeptidomics, Quantitative RT-PCR, Plaque Assay

Journal: PLoS Biology

Article Title: Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis

doi: 10.1371/journal.pbio.1000598

Figure Lengend Snippet: (A–C) Wild type BMDM or BMDM from IFNβ−/− knockout mice or from IFNAR1−/− knockout mice were mock treated, infected with mCMV, or treated with IFNβ (10 U/ml) for 24 h. RNA was collected and the gene expression of Hmgcs1 , Hmgcr , Idi1 , and Sqle was measured by qRT-PCR. Graphs show the level of expression of the indicated genes relative to mock-treated samples. Bars represent the mean ± SD of biological quadruplicates. (D) Wild type BMDM or BMDM from IFNAR1−/− knockout mice were infected with mCMV or treated with IFNβ (10 U/ml). After 48 h, free cholesterol concentration was measured by enzymatic assay . Bars represent the mean ± SD of biological quadruplicates. * p <0.05, ** p <0.01, *** p <0.001, determined with an unpaired Student's t test.

Article Snippet: Taqman Primer probe sets were purchased from Applied Biosystems, Warrington, UK (Assay ID: Hmgcs1: Mm00524111-m1; Hmgcr: Mm01282499-m1; Idi1: Mm00836417-g1; Sqle: Mm00436772-A1).

Techniques: Knock-Out, Infection, Gene Expression, Quantitative RT-PCR, Expressing, Concentration Assay, Enzymatic Assay

Journal: JID Innovations

Article Title: Bakuchiol and Ethyl (Linoleate/Oleate) Synergistically Modulate Endocannabinoid Tone in Keratinocytes and Repress Inflammatory Pathway mRNAs

doi: 10.1016/j.xjidi.2022.100178

Figure Lengend Snippet: RT-PCR analysis (replication study). ( a ) HMGCS1 . ( b ) MX1 . ( c ) OAS1 . A new set of KC samples was generated independently from those used in the original microarray study (n = 2 per treatment). Relative gene expression was evaluated using RT-PCR with GAPDH as a reference gene. The average normalized expression is shown for each treatment (±1 standard error). Treatments without the same letter differ significantly ( P < 0.05, Fisher's LSD) are shown. BAK, bakuchiol; CBD, cannabidiol; CTL, control; ELN, ethyl (linoleate/oleate); KC, keratinocyte; LSD, least significant difference.

Article Snippet: PCR primers were obtained from Thermo Fisher Scientific (TaqMan assays: HMGCS1 , Hs00940425_g1; MX1 , Hs00895604_m1; OAS1 , Hs00973635_m1; GAPDH , Hs02786624_g1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Generated, Microarray, Gene Expression, Expressing, Control