lpds  (Thermo Fisher)


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    Name:
    Penicillin Streptomycin Solution 10 000 units ea
    Description:
    The combination of the antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective action against gram positive and gram negative bacteria respectively Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacteria cell wall and indirectly by triggering the release of enzymes that further alter the cell wall Streptomycin was originally purified from Streptomyces griseus Streptomycin acts by binding to the 30S subunit of the bacterial ribosome leading to inhibition of protein synthesis and death in susceptible bacteria We offer a variety of antibiotics and antimycotics for cell culture applications This solution contains 10000 units of penicillin and 10000 µg of streptomycin per ml Product UseFor Research Use Only Not intended for animal or human diagnostic or therapeutic use
    Catalog Number:
    15140130
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
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    Structured Review

    Thermo Fisher lpds
    SA-β-Gal activity and immunofluorescence microscopy of LAMP1 for pseudoxanthoma elasticum (PXE) fibroblasts and normal human dermal fibroblasts (NHDF). Fibroblasts were cultivated for 72 h in medium with 10% fetal calf serum <t>(FCS)</t> or 10% lipoprotein-deficient fetal calf serum <t>(LPDS).</t> ( A ) Qualitative senescence assay for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Representative images are shown. Scale bar: 100 μm. ( B ) Quantitative senescence assay for PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/ PXE: *** p ≤ 0.001. 10% FCS/10% LPDS: + p ≤ 0.05; ns p > 0.05. ( C ) Immunofluorescence microscopy of LAMP1 (green) for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Cell nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar: 10 µm.
    The combination of the antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective action against gram positive and gram negative bacteria respectively Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacteria cell wall and indirectly by triggering the release of enzymes that further alter the cell wall Streptomycin was originally purified from Streptomyces griseus Streptomycin acts by binding to the 30S subunit of the bacterial ribosome leading to inhibition of protein synthesis and death in susceptible bacteria We offer a variety of antibiotics and antimycotics for cell culture applications This solution contains 10000 units of penicillin and 10000 µg of streptomycin per ml Product UseFor Research Use Only Not intended for animal or human diagnostic or therapeutic use
    https://www.bioz.com/result/lpds/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lpds - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Linking ABCC6 Deficiency in Primary Human Dermal Fibroblasts of PXE Patients to p21-Mediated Premature Cellular Senescence and the Development of a Proinflammatory Secretory Phenotype"

    Article Title: Linking ABCC6 Deficiency in Primary Human Dermal Fibroblasts of PXE Patients to p21-Mediated Premature Cellular Senescence and the Development of a Proinflammatory Secretory Phenotype

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21249665

    SA-β-Gal activity and immunofluorescence microscopy of LAMP1 for pseudoxanthoma elasticum (PXE) fibroblasts and normal human dermal fibroblasts (NHDF). Fibroblasts were cultivated for 72 h in medium with 10% fetal calf serum (FCS) or 10% lipoprotein-deficient fetal calf serum (LPDS). ( A ) Qualitative senescence assay for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Representative images are shown. Scale bar: 100 μm. ( B ) Quantitative senescence assay for PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/ PXE: *** p ≤ 0.001. 10% FCS/10% LPDS: + p ≤ 0.05; ns p > 0.05. ( C ) Immunofluorescence microscopy of LAMP1 (green) for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Cell nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar: 10 µm.
    Figure Legend Snippet: SA-β-Gal activity and immunofluorescence microscopy of LAMP1 for pseudoxanthoma elasticum (PXE) fibroblasts and normal human dermal fibroblasts (NHDF). Fibroblasts were cultivated for 72 h in medium with 10% fetal calf serum (FCS) or 10% lipoprotein-deficient fetal calf serum (LPDS). ( A ) Qualitative senescence assay for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Representative images are shown. Scale bar: 100 μm. ( B ) Quantitative senescence assay for PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/ PXE: *** p ≤ 0.001. 10% FCS/10% LPDS: + p ≤ 0.05; ns p > 0.05. ( C ) Immunofluorescence microscopy of LAMP1 (green) for PXE fibroblasts ( n = 3) and NHDF ( n = 3). Cell nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar: 10 µm.

    Techniques Used: Activity Assay, Immunofluorescence, Microscopy

    Relative p27, p21 und p53 mRNA-expression of PXE fibroblasts and NHDF. Fibroblasts were cultivated for 24 h in medium with 10% FCS or 10% LPDS. ( A ) Relative p27 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( B ) Relative p21 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( C ) Relative p53 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/ PXE: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05. 10% FCS/ 10% LPDS: + p ≤ 0.05; ## p ≤ 0.01; ns p > 0.05.
    Figure Legend Snippet: Relative p27, p21 und p53 mRNA-expression of PXE fibroblasts and NHDF. Fibroblasts were cultivated for 24 h in medium with 10% FCS or 10% LPDS. ( A ) Relative p27 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( B ) Relative p21 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( C ) Relative p53 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/ PXE: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05. 10% FCS/ 10% LPDS: + p ≤ 0.05; ## p ≤ 0.01; ns p > 0.05.

    Techniques Used: Expressing

    Relative mRNA-expression und protein expression of proinflammatory factors in PXE fibroblasts and NHDF. Fibroblasts were cultivated for 21 d in medium with 10% FCS or 10% LPDS. ( A ) Relative IL6 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( B ) IL6 protein concentration in supernatants of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( C ) Relative MCP1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( D ) MCP1 protein concentration in supernatants of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( E ) Relative ICAM1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/PXE: *** p ≤ 0.001; ns p > 0.05. 10% FCS/10% LPDS: # p ≤ 0.05; ns p > 0.05.
    Figure Legend Snippet: Relative mRNA-expression und protein expression of proinflammatory factors in PXE fibroblasts and NHDF. Fibroblasts were cultivated for 21 d in medium with 10% FCS or 10% LPDS. ( A ) Relative IL6 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( B ) IL6 protein concentration in supernatants of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( C ) Relative MCP1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( D ) MCP1 protein concentration in supernatants of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). ( E ) Relative ICAM1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3). Data are shown as mean ± SEM. Control/PXE: *** p ≤ 0.001; ns p > 0.05. 10% FCS/10% LPDS: # p ≤ 0.05; ns p > 0.05.

    Techniques Used: Expressing, Protein Concentration

    Relative LMNB1 mRNA-expression and immunofluorescence microscopy of lamin B1 for PXE fibroblasts and NHDF ( A ) Relative LMNB1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3) after 24 h cultivation in 10% FCS or 10% LPDS. Data are shown as mean ± SEM. Control/PXE: *** p ≤ 0.001; ns p > 0.05. 10% FCS/ 10% LPDS: ++ p ≤ 0.01; ns p > 0.05. ( B ) Immunofluorescence microscopy of lamin B1 (green) for PXE fibroblasts ( n = 3) and NHDF ( n = 3) after 72 h cultivation in 10% FCS or 10% LPDS. Cell nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar: 10 µm.
    Figure Legend Snippet: Relative LMNB1 mRNA-expression and immunofluorescence microscopy of lamin B1 for PXE fibroblasts and NHDF ( A ) Relative LMNB1 mRNA-expression of PXE fibroblasts (gray, n = 3) and NHDF (white, n = 3) after 24 h cultivation in 10% FCS or 10% LPDS. Data are shown as mean ± SEM. Control/PXE: *** p ≤ 0.001; ns p > 0.05. 10% FCS/ 10% LPDS: ++ p ≤ 0.01; ns p > 0.05. ( B ) Immunofluorescence microscopy of lamin B1 (green) for PXE fibroblasts ( n = 3) and NHDF ( n = 3) after 72 h cultivation in 10% FCS or 10% LPDS. Cell nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar: 10 µm.

    Techniques Used: Expressing, Immunofluorescence, Microscopy

    2) Product Images from "Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2 ▿"

    Article Title: Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2 ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00989-10

    PLD activity is required for Ras and ERK activation in response to cholesterol depletion. BHK cells were treated with lovastatin for 48 h in the presence of 10% LPDS. Control cells were cultured in 10% DCS. For the final 4 h of incubation,
    Figure Legend Snippet: PLD activity is required for Ras and ERK activation in response to cholesterol depletion. BHK cells were treated with lovastatin for 48 h in the presence of 10% LPDS. Control cells were cultured in 10% DCS. For the final 4 h of incubation,

    Techniques Used: Activity Assay, Activation Assay, Cell Culture, Incubation

    Therapeutic levels of lovastatin modulate ERK and Akt activation. BHK cells (a and e) and HUVECs (b and f) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h. Cell lysates
    Figure Legend Snippet: Therapeutic levels of lovastatin modulate ERK and Akt activation. BHK cells (a and e) and HUVECs (b and f) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h. Cell lysates

    Techniques Used: Activation Assay, Cell Culture

    Effect of therapeutic levels of lovastatin on cellular cholesterol and Ras prenylation. BHK cells (a) and HUVECs (c) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h.
    Figure Legend Snippet: Effect of therapeutic levels of lovastatin on cellular cholesterol and Ras prenylation. BHK cells (a) and HUVECs (c) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h.

    Techniques Used: Cell Culture

    EGF receptor expression is downregulated by lovastatin treatment. BHK cells were treated with lovastatin in the presence of 10% LPDS for 48 h. Control cells were cultured in standard growth medium containing 10% DCS. Total EGFR and phospho-EGFR
    Figure Legend Snippet: EGF receptor expression is downregulated by lovastatin treatment. BHK cells were treated with lovastatin in the presence of 10% LPDS for 48 h. Control cells were cultured in standard growth medium containing 10% DCS. Total EGFR and phospho-EGFR

    Techniques Used: Expressing, Cell Culture

    Lovastatin-induced EGFR downregulation is blocked by EGFR inhibition. BHK cells were cultured for 48 h in normal medium containing 10% DCS or 10% LPDS in the presence of lovastatin with or without 0.5 μM AG1478. Cell lysates were
    Figure Legend Snippet: Lovastatin-induced EGFR downregulation is blocked by EGFR inhibition. BHK cells were cultured for 48 h in normal medium containing 10% DCS or 10% LPDS in the presence of lovastatin with or without 0.5 μM AG1478. Cell lysates were

    Techniques Used: Inhibition, Cell Culture

    3) Product Images from "Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction"

    Article Title: Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0686-09.2009

    Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .
    Figure Legend Snippet: Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .

    Techniques Used: Transfection, Staining, Mutagenesis

    4) Product Images from "Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *"

    Article Title: Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.107854

    ATGL expression in macrophages and foam cells. Mouse peritoneal macrophages, human THP-1, and human monocyte-derived macrophages ( HMDM ) were cultivated in DMEM, 10% LPDS in the absence (control) or presence of 100 μg of acLDL/ml. Total RNA was isolated and reverse-transcribed, and mRNA expression of ATGL ( A and C ) and hormone-sensitive lipase ( HSL ) was determined by real time PCR, including murine hypoxanthine-guanine phosphoribosyltransferase or human ( hu ) porphobilinogen deaminase normalization ( A ). Untreated macrophages were arbitrarily set to 1. Data are expressed as mean values ( n = 3) ± S.E. of triplicate repeats. ***, p ≤ 0.001. B , cell extracts of macrophages, foam cells (40 μg per lane), and white adipose tissue ( WAT ) (10 and 40 μg per lane) were resolved by SDS-PAGE. Protein expression of ATGL and hormone-sensitive lipase were analyzed by Western blotting relative to the expression of β-actin. STD , standard.
    Figure Legend Snippet: ATGL expression in macrophages and foam cells. Mouse peritoneal macrophages, human THP-1, and human monocyte-derived macrophages ( HMDM ) were cultivated in DMEM, 10% LPDS in the absence (control) or presence of 100 μg of acLDL/ml. Total RNA was isolated and reverse-transcribed, and mRNA expression of ATGL ( A and C ) and hormone-sensitive lipase ( HSL ) was determined by real time PCR, including murine hypoxanthine-guanine phosphoribosyltransferase or human ( hu ) porphobilinogen deaminase normalization ( A ). Untreated macrophages were arbitrarily set to 1. Data are expressed as mean values ( n = 3) ± S.E. of triplicate repeats. ***, p ≤ 0.001. B , cell extracts of macrophages, foam cells (40 μg per lane), and white adipose tissue ( WAT ) (10 and 40 μg per lane) were resolved by SDS-PAGE. Protein expression of ATGL and hormone-sensitive lipase were analyzed by Western blotting relative to the expression of β-actin. STD , standard.

    Techniques Used: Expressing, Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

    mRNA levels of mitochondrial genes in Atgl −/− and WT macrophages and foam cells. MPM were cultivated in DMEM, 10% LPDS in the absence or presence of 100 μg of acLDL/ml (foam cells). Total RNA was isolated from cells and reverse-transcribed, and mRNA levels of carnitine palmitoyltransferase ( CPT )1a ( A ) and long chain acyl-CoA dehydrogenase ( LCAD ) ( B ) were determined by real time PCR, including normalization to hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) levels. Untreated MPM were arbitrarily set to 1. Data are expressed as mean values ± S.E. of triplicate repeats. C , long chain acyl-CoA. D , carnitine ester concentrations were determined in macrophages of Atgl −/− and WT mice. Data are expressed as mean values ( n = 4–5) ± S.E. correlated to protein concentrations. *, p
    Figure Legend Snippet: mRNA levels of mitochondrial genes in Atgl −/− and WT macrophages and foam cells. MPM were cultivated in DMEM, 10% LPDS in the absence or presence of 100 μg of acLDL/ml (foam cells). Total RNA was isolated from cells and reverse-transcribed, and mRNA levels of carnitine palmitoyltransferase ( CPT )1a ( A ) and long chain acyl-CoA dehydrogenase ( LCAD ) ( B ) were determined by real time PCR, including normalization to hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) levels. Untreated MPM were arbitrarily set to 1. Data are expressed as mean values ± S.E. of triplicate repeats. C , long chain acyl-CoA. D , carnitine ester concentrations were determined in macrophages of Atgl −/− and WT mice. Data are expressed as mean values ( n = 4–5) ± S.E. correlated to protein concentrations. *, p

    Techniques Used: Isolation, Cycling Probe Technology, Real-time Polymerase Chain Reaction, Mouse Assay

    Phagocytosis in Atgl −/− and WT macrophages. A , MPM from Atgl −/− and WT mice were cultivated in DMEM, 10% LPDS with 0, 6, and 25 m m glucose for 1 and 8 h, respectively. Phagocytosis of fluorescein-labeled E. coli particles is presented as mean values ( n = 6) ± S.E. of two independent experiments. Phagocytosis of WT cells was arbitrarily set to 100%. *, p
    Figure Legend Snippet: Phagocytosis in Atgl −/− and WT macrophages. A , MPM from Atgl −/− and WT mice were cultivated in DMEM, 10% LPDS with 0, 6, and 25 m m glucose for 1 and 8 h, respectively. Phagocytosis of fluorescein-labeled E. coli particles is presented as mean values ( n = 6) ± S.E. of two independent experiments. Phagocytosis of WT cells was arbitrarily set to 100%. *, p

    Techniques Used: Mouse Assay, Labeling

    ATP concentrations and ADP/ATP ratios in Atgl −/− and WT macrophages. A , ATP levels in Atgl −/− and WT MPM were measured after cultivating the cells for 24 h in DMEM, 10% LPDS containing 0, 6, and 25 m m glucose. Oligomycin (0.5 μ m ) was used as an ATP-depletion control. Data are presented as mean values ( n = 4) ± S.E. ***, p ≤ 0.001. B , ADP/ATP ratios in Atgl −/− and WT MPM were determined using the EnzyLight TM ADP/ATP ratio assay kit. Data are expressed as mean values ( n = 4) ± S.E. *, p
    Figure Legend Snippet: ATP concentrations and ADP/ATP ratios in Atgl −/− and WT macrophages. A , ATP levels in Atgl −/− and WT MPM were measured after cultivating the cells for 24 h in DMEM, 10% LPDS containing 0, 6, and 25 m m glucose. Oligomycin (0.5 μ m ) was used as an ATP-depletion control. Data are presented as mean values ( n = 4) ± S.E. ***, p ≤ 0.001. B , ADP/ATP ratios in Atgl −/− and WT MPM were determined using the EnzyLight TM ADP/ATP ratio assay kit. Data are expressed as mean values ( n = 4) ± S.E. *, p

    Techniques Used:

    5) Product Images from "Familial hypercholesterolemia class II low-density lipoprotein receptor response to statin treatment"

    Article Title: Familial hypercholesterolemia class II low-density lipoprotein receptor response to statin treatment

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.042911

    FH NC cells do not activate the UPR following rosuvastatin treatment. Following treatment with either carrier DM, RS, XS or tunicamycin (TM; 5 μg/ml for 4.5 h), cells were tested for ER stress and UPR markers at the mRNA level in iPSC/ESC and HLC. (A) qPCR analysis in iPSC indicated that Grp78 mRNA levels do not change with RS treatment compared to DM/XS controls across the three cell groups. TM significantly increased Grp78 mRNA in NC, C and H1 stem cells. (B) HLC presented the same trend, with TM significantly upregulating Grp78 transcripts, whereas RS did not. (C) PCR for spliced XBP1 ( SXBP1 ) visualized on a 2% agarose gel indicated that TM treatment induced splicing of XBP1 that was not present in RS-treated iPSC/ESC. Quantification of SXBP1/XBP1 shows significant SXBP1/XBP1 activation in TM-treated cells across all three cell types. (D) HLC also showed significant changes with treatment, and response to TM was significantly different from that to DM, RS and XS within cell lines, but no XBP1 cleavage was detected across the cell lines without TM treatment. (E) iPSC were treated with LPDS only, FBS only or LPDS with TM. qPCR quantification in iPSC indicated a Grp78 mRNA level pattern that is significantly different in response to TM treatment, but not in response to LPDS or FBS treatment, across the three cell groups. (F) PCR for SXBP1 indicated that TM treatment induced splicing of XBP1 that was not present in LPDS- or FBS-treated iPSC/ESC. Quantification of SXBP1/XBP1 shows significant SXBP1/XBP1 activation in TM-treated cells across the three cell types. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type of three experiments repeated in the laboratory using a two-way ANOVA Holm–Sidak post-hoc test. *P
    Figure Legend Snippet: FH NC cells do not activate the UPR following rosuvastatin treatment. Following treatment with either carrier DM, RS, XS or tunicamycin (TM; 5 μg/ml for 4.5 h), cells were tested for ER stress and UPR markers at the mRNA level in iPSC/ESC and HLC. (A) qPCR analysis in iPSC indicated that Grp78 mRNA levels do not change with RS treatment compared to DM/XS controls across the three cell groups. TM significantly increased Grp78 mRNA in NC, C and H1 stem cells. (B) HLC presented the same trend, with TM significantly upregulating Grp78 transcripts, whereas RS did not. (C) PCR for spliced XBP1 ( SXBP1 ) visualized on a 2% agarose gel indicated that TM treatment induced splicing of XBP1 that was not present in RS-treated iPSC/ESC. Quantification of SXBP1/XBP1 shows significant SXBP1/XBP1 activation in TM-treated cells across all three cell types. (D) HLC also showed significant changes with treatment, and response to TM was significantly different from that to DM, RS and XS within cell lines, but no XBP1 cleavage was detected across the cell lines without TM treatment. (E) iPSC were treated with LPDS only, FBS only or LPDS with TM. qPCR quantification in iPSC indicated a Grp78 mRNA level pattern that is significantly different in response to TM treatment, but not in response to LPDS or FBS treatment, across the three cell groups. (F) PCR for SXBP1 indicated that TM treatment induced splicing of XBP1 that was not present in LPDS- or FBS-treated iPSC/ESC. Quantification of SXBP1/XBP1 shows significant SXBP1/XBP1 activation in TM-treated cells across the three cell types. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type of three experiments repeated in the laboratory using a two-way ANOVA Holm–Sidak post-hoc test. *P

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Activation Assay

    LDLR-mediated cholesterol internalization is restored in C-HLC. (A) NC-HLC and C-HLC were treated overnight in 5% LPDS medium supplemented with rosuvastatin. This was followed by incubation with DiI-LDL for 6 h or 24 h. NC-HLC did not show any DiI fluorescence from LDL uptake until 24 h. C-HLC internalized DiI-LDL by 6 h, which increased after 24 h. Scale bars: 100 μm (each image has been linearly brightened to the same level). (B) HLC were imaged at 100× magnification for detection of DiI fluorescence localization. (C) HLC were treated with LPDS medium and rosuvastatin for 48 h followed by incubation in methyl-β-cyclodextrin (MBC) for 45 min when time point 0 h samples were collected. Cells were further incubated for 6 h or 24 h with unlabeled LDL then analyzed for cellular cholesterol concentration with respect to protein. NC-HLC showed no statistical difference in cellular cholesterol for all three time points, demonstrating the dysregulation of cholesterol metabolism in FH HLC. Corrected cells did not have statistically different cholesterol content after 6 h, demonstrating the effect of MBC treatment and plasma membrane cholesterol chelation on receptor-medicated endocytosis. After 24 h exposure to LDL-C, C-HLC were able to increase cellular cholesterol to a statistically significant level [61 µM/(µg/µl) compared to 11.6 µM/(µg/µl) at 0 h]. There was no difference in cholesterol concentration at 0 h between NC-HLC and C-HLC. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type and replicated three times in the laboratory. Statistics were performed using a two-way ANOVA Holm–Sidak post-hoc test. *P
    Figure Legend Snippet: LDLR-mediated cholesterol internalization is restored in C-HLC. (A) NC-HLC and C-HLC were treated overnight in 5% LPDS medium supplemented with rosuvastatin. This was followed by incubation with DiI-LDL for 6 h or 24 h. NC-HLC did not show any DiI fluorescence from LDL uptake until 24 h. C-HLC internalized DiI-LDL by 6 h, which increased after 24 h. Scale bars: 100 μm (each image has been linearly brightened to the same level). (B) HLC were imaged at 100× magnification for detection of DiI fluorescence localization. (C) HLC were treated with LPDS medium and rosuvastatin for 48 h followed by incubation in methyl-β-cyclodextrin (MBC) for 45 min when time point 0 h samples were collected. Cells were further incubated for 6 h or 24 h with unlabeled LDL then analyzed for cellular cholesterol concentration with respect to protein. NC-HLC showed no statistical difference in cellular cholesterol for all three time points, demonstrating the dysregulation of cholesterol metabolism in FH HLC. Corrected cells did not have statistically different cholesterol content after 6 h, demonstrating the effect of MBC treatment and plasma membrane cholesterol chelation on receptor-medicated endocytosis. After 24 h exposure to LDL-C, C-HLC were able to increase cellular cholesterol to a statistically significant level [61 µM/(µg/µl) compared to 11.6 µM/(µg/µl) at 0 h]. There was no difference in cholesterol concentration at 0 h between NC-HLC and C-HLC. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type and replicated three times in the laboratory. Statistics were performed using a two-way ANOVA Holm–Sidak post-hoc test. *P

    Techniques Used: Incubation, Fluorescence, Concentration Assay

    LDLR colocalizes with calnexin-ER in FH HLC. (A) After differentiation, HLC were treated overnight in LPDS medium supplemented with rosuvastatin followed by immunocytochemistry and confocal imaging, 100× oil immersion objective. AMIRA software was used to stack slices and merge channels to present an overview of localization of the LDLR (green), calnexin-ER (red) and nucleus (DAPI). (B) Quantification of colocalization using Fluoview software indicated that NC-HLC did have a significantly greater colocalization of the LDLR with calnexin than C-HLC. The graph values represent the mean±s.d. ( n =5) per cell type from three experiments repeated in the laboratory. Statistics were performed using an unpaired two-tailed Student's t -test, **P
    Figure Legend Snippet: LDLR colocalizes with calnexin-ER in FH HLC. (A) After differentiation, HLC were treated overnight in LPDS medium supplemented with rosuvastatin followed by immunocytochemistry and confocal imaging, 100× oil immersion objective. AMIRA software was used to stack slices and merge channels to present an overview of localization of the LDLR (green), calnexin-ER (red) and nucleus (DAPI). (B) Quantification of colocalization using Fluoview software indicated that NC-HLC did have a significantly greater colocalization of the LDLR with calnexin than C-HLC. The graph values represent the mean±s.d. ( n =5) per cell type from three experiments repeated in the laboratory. Statistics were performed using an unpaired two-tailed Student's t -test, **P

    Techniques Used: Immunocytochemistry, Imaging, Software, Two Tailed Test

    Rosuvastatin increases total LDLR protein levels and an accumulation of immature LDLR in NC cells. (A) PSC were treated overnight in LPDS medium supplemented with either lovastatin (Lova), rosuvastatin (RS) or excess sterols (XS). Western blot analysis for LDLR shows that NC-iPSC upregulate immature LDLR in Lova, whereas C-iPSC and H1-ESC express mature LDLR. RS treatment produces the same response, and LDLR is suppressed when exposed to sterols. Recombinant human LDLR protein (rhLDLR) was used as a detection control. (B) Quantification of total LDLR demonstrated that RS-treated NC-iPSC had a significantly greater total LDLR than C-iPSC and H1-ESC treated with RS. All three statin-treated cell types were significantly greater compared to their XS-treated counterparts. (C) iPSC/ESC were differentiated to HLC. Under the same conditions of rosuvastatin RS or XS treatment, NC-HLC express greater total LDLR, predominantly as immature protein, which is converted to all mature protein in C-HLC and H1-HLC. LDLR levels decreased with sterol treatment. (D) Two-way ANOVA analysis of total LDLR in HLC indicated a significant difference in mean between cell types and in response to RS treatment. Total LDLR in RS-treated NC cells was statistically different from that in RS-treated C and H1 cells. As expected, for each cell type, total LDLR was significantly different when comparing RS and XS treatments. (E) qPCR quantification of LDLR mRNA shows that RS treatment significantly increases LDLR transcript levels in NC-iPSC, C-iPSC and H1-ESC compared to DMSO (DM) control and XS treatment. (F) Similarly, HLC showed significant difference in mean LDLR mRNA with treatment and within cell type; in both C and H1 cells, means were different in response to RS treatment in comparison to DM and XS treatment, although no difference was detected within the NC cell treatment groups. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type of three experiments repeated in the laboratory using a two-way ANOVA Holm–Sidak post-hoc test. **P
    Figure Legend Snippet: Rosuvastatin increases total LDLR protein levels and an accumulation of immature LDLR in NC cells. (A) PSC were treated overnight in LPDS medium supplemented with either lovastatin (Lova), rosuvastatin (RS) or excess sterols (XS). Western blot analysis for LDLR shows that NC-iPSC upregulate immature LDLR in Lova, whereas C-iPSC and H1-ESC express mature LDLR. RS treatment produces the same response, and LDLR is suppressed when exposed to sterols. Recombinant human LDLR protein (rhLDLR) was used as a detection control. (B) Quantification of total LDLR demonstrated that RS-treated NC-iPSC had a significantly greater total LDLR than C-iPSC and H1-ESC treated with RS. All three statin-treated cell types were significantly greater compared to their XS-treated counterparts. (C) iPSC/ESC were differentiated to HLC. Under the same conditions of rosuvastatin RS or XS treatment, NC-HLC express greater total LDLR, predominantly as immature protein, which is converted to all mature protein in C-HLC and H1-HLC. LDLR levels decreased with sterol treatment. (D) Two-way ANOVA analysis of total LDLR in HLC indicated a significant difference in mean between cell types and in response to RS treatment. Total LDLR in RS-treated NC cells was statistically different from that in RS-treated C and H1 cells. As expected, for each cell type, total LDLR was significantly different when comparing RS and XS treatments. (E) qPCR quantification of LDLR mRNA shows that RS treatment significantly increases LDLR transcript levels in NC-iPSC, C-iPSC and H1-ESC compared to DMSO (DM) control and XS treatment. (F) Similarly, HLC showed significant difference in mean LDLR mRNA with treatment and within cell type; in both C and H1 cells, means were different in response to RS treatment in comparison to DM and XS treatment, although no difference was detected within the NC cell treatment groups. The graph values represent the mean±s.d. ( n =3) per treatment, per cell type of three experiments repeated in the laboratory using a two-way ANOVA Holm–Sidak post-hoc test. **P

    Techniques Used: Western Blot, Recombinant, Real-time Polymerase Chain Reaction

    Related Articles

    Cell Culture:

    Article Title: KSHV lytic mRNA is efficiently translated in the absence of eIF4F
    Article Snippet: .. All cells were maintained in 100 I/U of both penicillin and streptomycin. iSLK.219 cells were cultured with 10 mM puromycin (ThermoFisher Scientific) to maintain episome copy number of rKSHV.219. .. Expression of the RTA transgene and lytic reactivation in both the TREx-BCBL1-RTA and iSLK.219 was stimulated by treating the cells with 1 µg/mL doxycycline.

    Article Title: Sirtuin 1 Facilitates Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts through the miR-34a and p53 Pathways
    Article Snippet: Mouse Embryonic Stem Cell Culture and Differentiation Mouse embryonic stem cells (L4) were obtained from the Transgenic Core Facility, Department of Biochemistry, The University of Hong Kong. .. L4 and miPSC were cultured in mESC medium [DMEM with high glucose, 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco, Life Technologies), 0.1 mM MEM non-essential amino-acids (Gibco), sodium pyruvate (110 mg/L, Gibco), 50 µM beta-mercaptoethanol, 15% FBS (Gibco) and 1000 units/ml LIF (Millipore). ..

    Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma
    Article Snippet: Dendritic cells were then matured 72 h after culture using a maturation media consisting of human dendritic cell medium supplemented with 10 ng/mL IL-1β (Thermo Fisher Scientific, RIL1BI), 10 ng/mL 1L-6 (Thermo Fisher Scientific, RIL6I), 1 μg/mL PGE2 (TOCRIS, 2296) and 10 ng/mL TNF-α (Thermo Fisher Scientific, PHC3015) Dendritic cells were kept in maturation media for 16 h. Human CD8+ T Cells extracted from PBMCs were cultured for 16 h using a protocol adapted from a published protocol [ ] and media consisting of RPMI 1640 medium, 10% human serum albumin, 1% Penicillin/Streptomycin, 50 μM β-mercaptoethanol, 1× ITS (Thermo Fisher Scientific, 41400045), 0.15 μg/mL IL-2 (Thermofisher Scientific, RIL2I), and 0.05 ng/mL IL-7 (Thermo Fisher Scientific, RP-8645). .. MDSCs were cultured in 50% MDSC culture media (AIMV media containing 1% Penicillin/Streptomycin, 10 ng/mL IL-1β, 10 ng/mL 1L-6, 1 μg/mL PGE2), 2 ng/mL TGF-β1 (Thermo Fisher Scientific, 7754-BH-005/CF), 10 ng/mL TNF-α, 10 ng/mL VEGF (Thermo Fisher Scientific, RVEGFI), 10 ng/mL GM-CSF) and 50% conditioned medium collected from autologous organoid culture for 7 days, removing half of the media and replacing with fresh media/conditioned media every 48 h. ..

    Transfection:

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: .. The cells were induced 24 h after transfection with 100 nM DHB, in the presence of pen /strep (Gibco). ..

    Sequencing:

    Article Title: CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma
    Article Snippet: The PAK1 siRNA sequence was 5′-TTTCTTCTTAGGATCGCCCACACTC-3′ and negative control siRNA (control siRNA) for PAK1 was 5′- AGTCGACGTCAGCGAAGGC-3′ (Ambion, Austin, TX, USA). .. The PTP-PEST siRNA sequence was 5′-GGCAATTCCTCAGATATCA-3′ and negative control siRNA (control siRNA) for PTP-PEST was 5′- GGCAATTCCCCAGATATCA-3′ (Ambion, Austin, TX, USA). .. In vitro invasion assays The assay was performed using chambers with polycarbonate filters (8 μm pore size; Millipore).

    Negative Control:

    Article Title: CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma
    Article Snippet: The PAK1 siRNA sequence was 5′-TTTCTTCTTAGGATCGCCCACACTC-3′ and negative control siRNA (control siRNA) for PAK1 was 5′- AGTCGACGTCAGCGAAGGC-3′ (Ambion, Austin, TX, USA). .. The PTP-PEST siRNA sequence was 5′-GGCAATTCCTCAGATATCA-3′ and negative control siRNA (control siRNA) for PTP-PEST was 5′- GGCAATTCCCCAGATATCA-3′ (Ambion, Austin, TX, USA). .. In vitro invasion assays The assay was performed using chambers with polycarbonate filters (8 μm pore size; Millipore).

    Recombinant:

    Article Title: The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity
    Article Snippet: Cell culture and transfectionU2Os cells (obtained from ATCC, CA) were maintained in 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) (ThermoFisher Scientific, CA, Cat# 11995–065) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific, Cat# 12483–020) and 1% penicillin-streptomycin (ThermoFisher Scientific, Cat# 15140122). .. Mono-Mac-6 (MM6) cells (obtained from DSMZ, Cat# ACC 124) were maintained in RPMI 1640 (ThermoFisher Scientific) supplemented with 10% FBS, 1% penicillin-streptavidin, 1% MEM non-essential amino acids (ThermoFisher Scientific, Cat# 11140076) and 10 µg/ml recombinant human insulin (Sigma Aldrich, Cat# 91077C). .. Mouse mammary 66cl4 cells were obtained from Dr. Josie Ursini-Siegel (Lady Davis Institute, Montreal, QC, Canada) and cultured in RPMI 1640 (ThermoFisher Scientific, Cat# 22400–089) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin.

    Over Expression:

    Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts
    Article Snippet: .. Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.). .. Subconfluent PTP-PEST (−/−) cells were transfected with this recombinant vector using Lipofectamine Plus ( GIBCO BRL ).

    Plasmid Preparation:

    Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts
    Article Snippet: .. Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.). .. Subconfluent PTP-PEST (−/−) cells were transfected with this recombinant vector using Lipofectamine Plus ( GIBCO BRL ).

    Marker:

    Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts
    Article Snippet: .. Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.). .. Subconfluent PTP-PEST (−/−) cells were transfected with this recombinant vector using Lipofectamine Plus ( GIBCO BRL ).

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  • 99
    Thermo Fisher dmem medium
    25HC does not directly affect bacterial infectivity or host viability. 25HC could inhibit L. monocytogenes infection through different mechanisms. For example, it may (1) directly reduce bacterial viability, (2) inhibit the expression or function of bacterial virulence factors, (3) induce host cell death, or (4) regulate host cellular processes that limit bacterial infection. a , To determine whether 25HC directly reduced bacterial viability, a starting bacterial culture was back-diluted in <t>DMEM</t> (10% FBS) supplemented with vehicle (EtOH) or 25HC (5 μM). Bacterial cultures were incubated at 37°C while shaking at 200 rpm, and OD 600 was measured for each sample at the indicated time points. Mean values from 3 independent experiments are plotted, and error bars show s.d. b , To determine whether 25HC directly modifies bacterial virulence, GFP-expressing L. monocytogenes were cultured overnight in BHI supplemented with vehicle (EtOH) or 25HC (5 μM) and <t>HEK293A</t> cells were then infected with bacteria from either culture (MOI = 20, 6 hours). Infection was analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). c-d , Host cell viability was assessed in cells treated with 25HC (c) or in cells virally transduced with CH25H (d). HEK293A cells were treated with 25HC (5 μM) or vehicle (EtOH) for 6, 16, or 24 hours (c) or transduced with Fluc or CH25H for 72 hours (d). Cell viability was evaluated by measuring ATP production using CellTiter-Glo assays. Data are normalized to vehicle (EtOH) in (c), and Fluc in (d). Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by student’s unpaired t-test (two-tailed).
    Dmem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lpds
    ATGL expression in macrophages and foam cells. Mouse peritoneal macrophages, human THP-1, and human monocyte-derived macrophages ( HMDM ) were cultivated in <t>DMEM,</t> 10% <t>LPDS</t> in the absence (control) or presence of 100 μg of acLDL/ml. Total RNA was isolated and reverse-transcribed, and mRNA expression of ATGL ( A and C ) and hormone-sensitive lipase ( HSL ) was determined by real time PCR, including murine hypoxanthine-guanine phosphoribosyltransferase or human ( hu ) porphobilinogen deaminase normalization ( A ). Untreated macrophages were arbitrarily set to 1. Data are expressed as mean values ( n = 3) ± S.E. of triplicate repeats. ***, p ≤ 0.001. B , cell extracts of macrophages, foam cells (40 μg per lane), and white adipose tissue ( WAT ) (10 and 40 μg per lane) were resolved by SDS-PAGE. Protein expression of ATGL and hormone-sensitive lipase were analyzed by Western blotting relative to the expression of β-actin. STD , standard.
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    Thermo Fisher lipoprotein depleted serum lpds
    Sesn1 regulates cellular and plasma cholesterol content independent of LDLR and SREBP2 signaling. (A) Liver PCSK9 and LDLR protein levels in mice after 0.2% cholesterol diet feeding for three days. n = 4. (B) Plasma total cholesterol levels in Ldlr −/− Sesn1 +/+ (black) and Ldlr −/− Sesn1 +/− (red) male mice after 4 and 12 weeks of cholesterol diet feeding. n > 9. (C) Liver mRNA expression levels of cholesterol and lipid genes in male Sesn1 +/+ (black) and Sesn1 −/− (red) mice fed a cholesterol diet for three days. n = 4. (D) Cellular cholesterol levels in <t>AML12</t> cells after Sesn1 silencing (red) or control (black) incubated in FBS or lipoprotein depleted serum <t>(LPDS)</t> medium. n = 4. (E) LDLR protein levels in Sesn1 siRNA or control siRNA treated AML12 cells with FBS, n = 4. (F) mRNA expression levels of cholesterol and lipid metabolism genes in AML12 cells after Sesn1 silencing with FBS or LPDS medium. n = 3. Data represented is mean ± SE, ** P
    Lipoprotein Depleted Serum Lpds, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher lpd specific raph1 small interfering rna sirna
    Depletion of <t>Lpd</t> in HeLa cells using <t>RAPH1</t> <t>siRNA</t> interference significantly reduced the number of plaques formed by L. monocytogenes . The results of Western blot analysis using anti-Lpd antibody against the cell lysate of HeLa cells either transformed with Lpd-expressing plasmid or transfected with 1 nM nonspecific Luc siRNA as a control with RAPH1 individual or mixed siRNAs for 3 days are shown. Tubulin was used as the control. The relative intensity of the band of each sample against HeLa cell lysate was measured using ImageJ. The number of plaques was significantly lower in HeLa cell knockdown of Lpd expression using a RAPH1 siRNA mixture and three of the individual siRNAs. Error bars indicate standard errors. Stars indicate statistically significant differences. *, P
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    25HC does not directly affect bacterial infectivity or host viability. 25HC could inhibit L. monocytogenes infection through different mechanisms. For example, it may (1) directly reduce bacterial viability, (2) inhibit the expression or function of bacterial virulence factors, (3) induce host cell death, or (4) regulate host cellular processes that limit bacterial infection. a , To determine whether 25HC directly reduced bacterial viability, a starting bacterial culture was back-diluted in DMEM (10% FBS) supplemented with vehicle (EtOH) or 25HC (5 μM). Bacterial cultures were incubated at 37°C while shaking at 200 rpm, and OD 600 was measured for each sample at the indicated time points. Mean values from 3 independent experiments are plotted, and error bars show s.d. b , To determine whether 25HC directly modifies bacterial virulence, GFP-expressing L. monocytogenes were cultured overnight in BHI supplemented with vehicle (EtOH) or 25HC (5 μM) and HEK293A cells were then infected with bacteria from either culture (MOI = 20, 6 hours). Infection was analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). c-d , Host cell viability was assessed in cells treated with 25HC (c) or in cells virally transduced with CH25H (d). HEK293A cells were treated with 25HC (5 μM) or vehicle (EtOH) for 6, 16, or 24 hours (c) or transduced with Fluc or CH25H for 72 hours (d). Cell viability was evaluated by measuring ATP production using CellTiter-Glo assays. Data are normalized to vehicle (EtOH) in (c), and Fluc in (d). Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by student’s unpaired t-test (two-tailed).

    Journal: Nature microbiology

    Article Title: Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol

    doi: 10.1038/s41564-020-0701-5

    Figure Lengend Snippet: 25HC does not directly affect bacterial infectivity or host viability. 25HC could inhibit L. monocytogenes infection through different mechanisms. For example, it may (1) directly reduce bacterial viability, (2) inhibit the expression or function of bacterial virulence factors, (3) induce host cell death, or (4) regulate host cellular processes that limit bacterial infection. a , To determine whether 25HC directly reduced bacterial viability, a starting bacterial culture was back-diluted in DMEM (10% FBS) supplemented with vehicle (EtOH) or 25HC (5 μM). Bacterial cultures were incubated at 37°C while shaking at 200 rpm, and OD 600 was measured for each sample at the indicated time points. Mean values from 3 independent experiments are plotted, and error bars show s.d. b , To determine whether 25HC directly modifies bacterial virulence, GFP-expressing L. monocytogenes were cultured overnight in BHI supplemented with vehicle (EtOH) or 25HC (5 μM) and HEK293A cells were then infected with bacteria from either culture (MOI = 20, 6 hours). Infection was analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). c-d , Host cell viability was assessed in cells treated with 25HC (c) or in cells virally transduced with CH25H (d). HEK293A cells were treated with 25HC (5 μM) or vehicle (EtOH) for 6, 16, or 24 hours (c) or transduced with Fluc or CH25H for 72 hours (d). Cell viability was evaluated by measuring ATP production using CellTiter-Glo assays. Data are normalized to vehicle (EtOH) in (c), and Fluc in (d). Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by student’s unpaired t-test (two-tailed).

    Article Snippet: For the BMDM medium-transfer infection assays ( , and ), 15,000 HEK293A cells were seeded per well of a 96-well plate in DMEM medium (containing 5% LPDS, 1×NEAA).

    Techniques: Infection, Expressing, Incubation, Cell Culture, Flow Cytometry, Two Tailed Test, Transduction

    ATGL expression in macrophages and foam cells. Mouse peritoneal macrophages, human THP-1, and human monocyte-derived macrophages ( HMDM ) were cultivated in DMEM, 10% LPDS in the absence (control) or presence of 100 μg of acLDL/ml. Total RNA was isolated and reverse-transcribed, and mRNA expression of ATGL ( A and C ) and hormone-sensitive lipase ( HSL ) was determined by real time PCR, including murine hypoxanthine-guanine phosphoribosyltransferase or human ( hu ) porphobilinogen deaminase normalization ( A ). Untreated macrophages were arbitrarily set to 1. Data are expressed as mean values ( n = 3) ± S.E. of triplicate repeats. ***, p ≤ 0.001. B , cell extracts of macrophages, foam cells (40 μg per lane), and white adipose tissue ( WAT ) (10 and 40 μg per lane) were resolved by SDS-PAGE. Protein expression of ATGL and hormone-sensitive lipase were analyzed by Western blotting relative to the expression of β-actin. STD , standard.

    Journal: The Journal of Biological Chemistry

    Article Title: Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *

    doi: 10.1074/jbc.M110.107854

    Figure Lengend Snippet: ATGL expression in macrophages and foam cells. Mouse peritoneal macrophages, human THP-1, and human monocyte-derived macrophages ( HMDM ) were cultivated in DMEM, 10% LPDS in the absence (control) or presence of 100 μg of acLDL/ml. Total RNA was isolated and reverse-transcribed, and mRNA expression of ATGL ( A and C ) and hormone-sensitive lipase ( HSL ) was determined by real time PCR, including murine hypoxanthine-guanine phosphoribosyltransferase or human ( hu ) porphobilinogen deaminase normalization ( A ). Untreated macrophages were arbitrarily set to 1. Data are expressed as mean values ( n = 3) ± S.E. of triplicate repeats. ***, p ≤ 0.001. B , cell extracts of macrophages, foam cells (40 μg per lane), and white adipose tissue ( WAT ) (10 and 40 μg per lane) were resolved by SDS-PAGE. Protein expression of ATGL and hormone-sensitive lipase were analyzed by Western blotting relative to the expression of β-actin. STD , standard.

    Article Snippet: Macrophages were cultured in DMEM, 10% LPDS (Invitrogen) supplemented with 1% penicillin, 1% streptomycin for 2 h. Thereafter, cells were washed twice with PBS, and if not stated otherwise, the adherent macrophages were cultured in high glucose (25 mm ) DMEM containing 4 mm glutamine, 1 mm pyruvate, and 10% LPDS.

    Techniques: Expressing, Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

    mRNA levels of mitochondrial genes in Atgl −/− and WT macrophages and foam cells. MPM were cultivated in DMEM, 10% LPDS in the absence or presence of 100 μg of acLDL/ml (foam cells). Total RNA was isolated from cells and reverse-transcribed, and mRNA levels of carnitine palmitoyltransferase ( CPT )1a ( A ) and long chain acyl-CoA dehydrogenase ( LCAD ) ( B ) were determined by real time PCR, including normalization to hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) levels. Untreated MPM were arbitrarily set to 1. Data are expressed as mean values ± S.E. of triplicate repeats. C , long chain acyl-CoA. D , carnitine ester concentrations were determined in macrophages of Atgl −/− and WT mice. Data are expressed as mean values ( n = 4–5) ± S.E. correlated to protein concentrations. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *

    doi: 10.1074/jbc.M110.107854

    Figure Lengend Snippet: mRNA levels of mitochondrial genes in Atgl −/− and WT macrophages and foam cells. MPM were cultivated in DMEM, 10% LPDS in the absence or presence of 100 μg of acLDL/ml (foam cells). Total RNA was isolated from cells and reverse-transcribed, and mRNA levels of carnitine palmitoyltransferase ( CPT )1a ( A ) and long chain acyl-CoA dehydrogenase ( LCAD ) ( B ) were determined by real time PCR, including normalization to hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) levels. Untreated MPM were arbitrarily set to 1. Data are expressed as mean values ± S.E. of triplicate repeats. C , long chain acyl-CoA. D , carnitine ester concentrations were determined in macrophages of Atgl −/− and WT mice. Data are expressed as mean values ( n = 4–5) ± S.E. correlated to protein concentrations. *, p

    Article Snippet: Macrophages were cultured in DMEM, 10% LPDS (Invitrogen) supplemented with 1% penicillin, 1% streptomycin for 2 h. Thereafter, cells were washed twice with PBS, and if not stated otherwise, the adherent macrophages were cultured in high glucose (25 mm ) DMEM containing 4 mm glutamine, 1 mm pyruvate, and 10% LPDS.

    Techniques: Isolation, Cycling Probe Technology, Real-time Polymerase Chain Reaction, Mouse Assay

    Phagocytosis in Atgl −/− and WT macrophages. A , MPM from Atgl −/− and WT mice were cultivated in DMEM, 10% LPDS with 0, 6, and 25 m m glucose for 1 and 8 h, respectively. Phagocytosis of fluorescein-labeled E. coli particles is presented as mean values ( n = 6) ± S.E. of two independent experiments. Phagocytosis of WT cells was arbitrarily set to 100%. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *

    doi: 10.1074/jbc.M110.107854

    Figure Lengend Snippet: Phagocytosis in Atgl −/− and WT macrophages. A , MPM from Atgl −/− and WT mice were cultivated in DMEM, 10% LPDS with 0, 6, and 25 m m glucose for 1 and 8 h, respectively. Phagocytosis of fluorescein-labeled E. coli particles is presented as mean values ( n = 6) ± S.E. of two independent experiments. Phagocytosis of WT cells was arbitrarily set to 100%. *, p

    Article Snippet: Macrophages were cultured in DMEM, 10% LPDS (Invitrogen) supplemented with 1% penicillin, 1% streptomycin for 2 h. Thereafter, cells were washed twice with PBS, and if not stated otherwise, the adherent macrophages were cultured in high glucose (25 mm ) DMEM containing 4 mm glutamine, 1 mm pyruvate, and 10% LPDS.

    Techniques: Mouse Assay, Labeling

    ATP concentrations and ADP/ATP ratios in Atgl −/− and WT macrophages. A , ATP levels in Atgl −/− and WT MPM were measured after cultivating the cells for 24 h in DMEM, 10% LPDS containing 0, 6, and 25 m m glucose. Oligomycin (0.5 μ m ) was used as an ATP-depletion control. Data are presented as mean values ( n = 4) ± S.E. ***, p ≤ 0.001. B , ADP/ATP ratios in Atgl −/− and WT MPM were determined using the EnzyLight TM ADP/ATP ratio assay kit. Data are expressed as mean values ( n = 4) ± S.E. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase *

    doi: 10.1074/jbc.M110.107854

    Figure Lengend Snippet: ATP concentrations and ADP/ATP ratios in Atgl −/− and WT macrophages. A , ATP levels in Atgl −/− and WT MPM were measured after cultivating the cells for 24 h in DMEM, 10% LPDS containing 0, 6, and 25 m m glucose. Oligomycin (0.5 μ m ) was used as an ATP-depletion control. Data are presented as mean values ( n = 4) ± S.E. ***, p ≤ 0.001. B , ADP/ATP ratios in Atgl −/− and WT MPM were determined using the EnzyLight TM ADP/ATP ratio assay kit. Data are expressed as mean values ( n = 4) ± S.E. *, p

    Article Snippet: Macrophages were cultured in DMEM, 10% LPDS (Invitrogen) supplemented with 1% penicillin, 1% streptomycin for 2 h. Thereafter, cells were washed twice with PBS, and if not stated otherwise, the adherent macrophages were cultured in high glucose (25 mm ) DMEM containing 4 mm glutamine, 1 mm pyruvate, and 10% LPDS.

    Techniques:

    Sesn1 regulates cellular and plasma cholesterol content independent of LDLR and SREBP2 signaling. (A) Liver PCSK9 and LDLR protein levels in mice after 0.2% cholesterol diet feeding for three days. n = 4. (B) Plasma total cholesterol levels in Ldlr −/− Sesn1 +/+ (black) and Ldlr −/− Sesn1 +/− (red) male mice after 4 and 12 weeks of cholesterol diet feeding. n > 9. (C) Liver mRNA expression levels of cholesterol and lipid genes in male Sesn1 +/+ (black) and Sesn1 −/− (red) mice fed a cholesterol diet for three days. n = 4. (D) Cellular cholesterol levels in AML12 cells after Sesn1 silencing (red) or control (black) incubated in FBS or lipoprotein depleted serum (LPDS) medium. n = 4. (E) LDLR protein levels in Sesn1 siRNA or control siRNA treated AML12 cells with FBS, n = 4. (F) mRNA expression levels of cholesterol and lipid metabolism genes in AML12 cells after Sesn1 silencing with FBS or LPDS medium. n = 3. Data represented is mean ± SE, ** P

    Journal: Cell metabolism

    Article Title: Integrating mouse and human genetic data to move beyond GWAS and identify causal genes in cholesterol metabolism

    doi: 10.1016/j.cmet.2020.02.015

    Figure Lengend Snippet: Sesn1 regulates cellular and plasma cholesterol content independent of LDLR and SREBP2 signaling. (A) Liver PCSK9 and LDLR protein levels in mice after 0.2% cholesterol diet feeding for three days. n = 4. (B) Plasma total cholesterol levels in Ldlr −/− Sesn1 +/+ (black) and Ldlr −/− Sesn1 +/− (red) male mice after 4 and 12 weeks of cholesterol diet feeding. n > 9. (C) Liver mRNA expression levels of cholesterol and lipid genes in male Sesn1 +/+ (black) and Sesn1 −/− (red) mice fed a cholesterol diet for three days. n = 4. (D) Cellular cholesterol levels in AML12 cells after Sesn1 silencing (red) or control (black) incubated in FBS or lipoprotein depleted serum (LPDS) medium. n = 4. (E) LDLR protein levels in Sesn1 siRNA or control siRNA treated AML12 cells with FBS, n = 4. (F) mRNA expression levels of cholesterol and lipid metabolism genes in AML12 cells after Sesn1 silencing with FBS or LPDS medium. n = 3. Data represented is mean ± SE, ** P

    Article Snippet: AML12 cells were incubated with lipoprotein depleted serum (LPDS) (Alfa Aesar, Havervill, MA) medium for additional 24 hours before harvest.

    Techniques: Mouse Assay, Expressing, Incubation

    Depletion of Lpd in HeLa cells using RAPH1 siRNA interference significantly reduced the number of plaques formed by L. monocytogenes . The results of Western blot analysis using anti-Lpd antibody against the cell lysate of HeLa cells either transformed with Lpd-expressing plasmid or transfected with 1 nM nonspecific Luc siRNA as a control with RAPH1 individual or mixed siRNAs for 3 days are shown. Tubulin was used as the control. The relative intensity of the band of each sample against HeLa cell lysate was measured using ImageJ. The number of plaques was significantly lower in HeLa cell knockdown of Lpd expression using a RAPH1 siRNA mixture and three of the individual siRNAs. Error bars indicate standard errors. Stars indicate statistically significant differences. *, P

    Journal: Infection and Immunity

    Article Title: Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes

    doi: 10.1128/IAI.00193-15

    Figure Lengend Snippet: Depletion of Lpd in HeLa cells using RAPH1 siRNA interference significantly reduced the number of plaques formed by L. monocytogenes . The results of Western blot analysis using anti-Lpd antibody against the cell lysate of HeLa cells either transformed with Lpd-expressing plasmid or transfected with 1 nM nonspecific Luc siRNA as a control with RAPH1 individual or mixed siRNAs for 3 days are shown. Tubulin was used as the control. The relative intensity of the band of each sample against HeLa cell lysate was measured using ImageJ. The number of plaques was significantly lower in HeLa cell knockdown of Lpd expression using a RAPH1 siRNA mixture and three of the individual siRNAs. Error bars indicate standard errors. Stars indicate statistically significant differences. *, P

    Article Snippet: Lpd RNA interference (RNAi) was performed using Interferin (Polyplus)-mediated delivery of Lpd-specific RAPH1 small interfering RNA (siRNA) (Thermo Scientific).

    Techniques: Western Blot, Transformation Assay, Expressing, Plasmid Preparation, Transfection