Journal: BMC Genomics

Article Title: Transcriptome profiling revealed early vascular smooth muscle cell gene activation following focal ischemic stroke in female rats – comparisons with males

doi: 10.1186/s12864-020-07295-2

Figure Lengend Snippet: Further analysis of the upregulated gene targets in the female rat middle cerebral arteries (MCAs). a . Heatmap and dendrograms illustrating hierarchical clustering of the expression of the gene targets for the experimental groups, occluded MCAs (R), non-occluded MCAs (L) and control MCAs (C), based on outcomes from the Affymetrix whole-transcriptome expression profiling (microarray analysis). The highest gene expression of ADAM metallopeptidase with thrombospondin type 1 motif 4 ( Adamts4 ), C-C motif chemokine 2 ( Ccl2 ), Fos-like antigen 1 ( Fosl1 ), Jun B proto-oncogene ( JunB ), oxidized low density lipoprotein receptor 1 ( Olr1 ), sphingosine-1-phosphate receptor 3 ( S1pr3 ), serpin peptidase inhibitor, clade E member 1 ( Serpine1 ) and suppressor of cytokine signaling 3 ( Socs3 ) were observed for the occluded MCAs. b . Dot plot showing categorization of the gene targets, Adamts4 , Ccl2 , Fosl1 , JunB , Olr1 , S1pr3 , Serpine1 and Socs3 , into selected GO terms within the biological process domain. All gene targets were annotated to response to stress

Article Snippet: Taqman gene expression assays for Ccl2 (Rn00580555_m1), Olr1 (Rn00591116_m1), Adamts4 (Rn02103282_s1), Serpine1 (Rn01481341_m1), S1pr3 (Rn01757498_m1), Socs3 (Rn01470502_g1), JunB (Rn00572994_s1) and Fosl1 (Rn00564119_m1) were purchased from ThermoFisher Scientific, MA, USA.

Techniques: Expressing, Control, Microarray, Gene Expression

Journal: BMC Genomics

Article Title: Transcriptome profiling revealed early vascular smooth muscle cell gene activation following focal ischemic stroke in female rats – comparisons with males

doi: 10.1186/s12864-020-07295-2

Figure Lengend Snippet: Validation of gene targets in male and female middle cerebral arteries (MCAs) after tMCAO. Differential gene expression of the selected upregulated gene targets was validated by qPCR. When comparing delta cycle threshold (dCT) values between the (non-operated) control MCAs, and the non-occluded and occluded MCAs after tMCAO [transient middle cerebral artery occlusion], significant elevations were seen in both sexes for C-C motif chemokine 2 ( Ccl2 ), oxidized low-density lipoprotein receptor 1 ( Olr1 ), a disintegrin and metalloproteinase with thrombospondin type 1 motif, 4 ( Adamts4 ), and serine protease inhibitor, clade E, member 1 ( Serpine1 , one outlier excluded for this gene dataset). Sex did not significantly affect the outcome for any of the genes being validated ( p = 0.22–0.52) therefore, only the fixed effect 'experimental group' was fitted into the model ( p < 0.01). First, we compared the occluded and non-occluded MCAs to the reference ‘control MCAs’, and second, we compared the occluded MCAs to the reference ‘non-occluded MCAs’. Information within parentheses shows number of samples for the sex (F/M) and group in question. Statistical tests were performed (see methods): * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 and NS indicates non-significance ( p > 0.05). Abbreviations: F, female; M, Male

Article Snippet: Taqman gene expression assays for Ccl2 (Rn00580555_m1), Olr1 (Rn00591116_m1), Adamts4 (Rn02103282_s1), Serpine1 (Rn01481341_m1), S1pr3 (Rn01757498_m1), Socs3 (Rn01470502_g1), JunB (Rn00572994_s1) and Fosl1 (Rn00564119_m1) were purchased from ThermoFisher Scientific, MA, USA.

Techniques: Biomarker Discovery, Gene Expression, Control, Protease Inhibitor

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Low-density lipoprotein (LDLR) protein expression and 19-dioctadecyl-3,3,39,39-tetramethyl indocarbocyanine (DiI)-low-density lipoprotein (LDL) uptake in TPC1 and BCPAP cell lines. ( A ) Cells were treated with basal conditions (5% FBS) or LDL (200 μg/mL ApoB) and harvested at 24 h, 48 h and 72 h before analysis. Left panel: one representative blot is shown, and stain-free gel (SF) was used as the loading control. Graphs show densitometry of the Western blots relative to basal condition-treated cells (5% FBS) in comparison to LDL-treated cells. ( B ) Cells were exposed to DiI-LDL (200 μg/mL ApoB) for 24 h, 48 h and 72 h before analysis for mean fluorescence intensity by fluorescence spectrometer to compare LDL uptake between both cell lines. Statistical analysis: a two-way ANOVA test plus Sidak’s multiple comparisons test were performed to compare the LDLR protein expression and DiI-LDL uptake between the two groups at each time point (* p < 0.05, ** p < 0.002, *** p = 0.0003, **** p < 0.001). Data are expressed as mean ± SEM of a minimum of three independent experiments (N = 3).

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Expressing, Staining, Control, Western Blot, Comparison, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Confocal images of DiI-LDL uptake and lysosome quantification. ( A ) Confocal microscopy of Hoechst-stained (blue), DiI-LDL-stained (green) and lysosome-GFP (red) in TPC1 and BCPAP cell lines. Cells were treated for 24 h, 48 h and 72 h with DiI-LDL (200 μg/mL ApoB). The presence of colocalization of the red and green signals in the merged images is highlighted in yellow. The scale bar size was set to 50 µm. ( B ) Quantity of lysosomes in TPC1 and BCPAP cell lines after DiI-LDL (200 μg/mL ApoB) incubation for 24 h, 48 h and 72 h. Statistical analysis: a two-way ANOVA test plus Sidak’s multiple comparisons test were performed to compare the lysosome quantification between the cell lines (*** p < 0.0009 and **** p < 0.0001). Data are expressed as mean ± SEM of three independent experiments (N = 3).

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Confocal Microscopy, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Percentage of cellular proliferation determined by MTT assay of the TPC1 and BCPAP cell lines. Cells were treated with basal conditions (5% FBS), as a control, or with LDL (200 μg/mL ApoB) for 24 h, 48 h and 72 h. Statistical analysis: a two-way ANOVA test plus Sidak’s multiple comparisons test were performed to compare both groups. (*** p < 0.0009, **** p < 0.001). Data are expressed as mean ± SEM of three independent experiments (N = 3) carried out in quintuplicate.

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: MTT Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Effects of LDL on cellular migration. A line was scratched in both the TPC1 and BCPAP cell lines, and cultures were treated with 5% FBS, as a control, or with LDL (200 µg/mL ApoB) for 16 h. ( A ) The graph represents the percentage of wound-healing repair (**** p < 0.0001) in the TPC1 cell line treated with LDL (200 µg/mL ApoB) compared to the control at basal conditions (5% FBS). ( B ) The graph represents the percentage of suspension BCPAP cells treated with LDL (200 µg/mL ApoB) compared to the control at basal conditions (5% FBS). Images are at 10× resolution. Statistical analysis: an unpaired t -test was performed to compare the LDL-treated cells with the control condition (*** p = 0.0009, **** p < 0.0001). The results are presented as the mean ± SEM of three independent experiments.

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Migration, Control, Suspension

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Western blot analysis for PI3K–AKT–MTOR and RAS/RAF/MAPK (MEK)/ERK pathways. ( A ) TPC1 cells were treated with basal conditions (5% FBS) or LDL (200 μg/mL ApoB) and harvested at 24 h, 48 h and 72 h. Last panel: representatives blots are shown. Stain-free (SF) gel was used as the loading control. Graphs show the densitometry of the Western blots relative to basal condition-treated cells in comparison to LDL-treated cells. ( B ) BCPAP cells were treated with basal conditions (5% FBS) or LDL (200 μg/mL ApoB) and harvested at 24 h, 48 h and 72 h. Last panel: representative blots are shown. Stain-free (SF) gel was used as the loading control. Graphs show densitometry of the Western blots relative to basal condition-treated cells in comparison to LDL-treated cells. Statistical analysis: two-way ANOVA test plus Sidak’s multiple comparisons test (* p < 0.01, ** p < 0.001). Data are expressed as mean ± SEM of a minimum of three independent experiments (N = 3).

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Western Blot, Staining, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Western blot analysis of Ras/Raf/MAPK (MEK)/ERK pathway and cellular proliferation determined by MTT assay of BCPAP cell line after LDL (200 µg/mL ApoB) incubation and/or vemurafenib treatment (1 µM) compared to the LDL-only treatment condition (200 µg/mL ApoB + DMSO 0.1%) for 24 h, 48 h and 72 h. ( A ) Representative blots of LDLR, ERK and p-ERK are shown. Stain-free (SF) gel was used as the loading control. ( B ) Graphs show the densitometry analysis of the Western blots of LDLR, ERK and p-ERK after LDL (200 µg/mL ApoB) incubation and/or vemurafenib treatment (1 μM) for 24 h, 48 h and 72 h in comparison to the LDL-only treatment condition (200 µg/mL ApoB + DMSO 0.1%). ( C ) Proliferation percentage determined by MTT assay in the BCPAP cell line after incubation with LDL (200 µg/mL ApoB) and/or vemurafenib treatment (1 µM) compared to the LDL-only treatment condition (200 µg/mL ApoB + DMSO 0.1%) at 24 h, 48 h and 72 h. Statistical analysis: one-way ANOVA test plus Tukey’s multiple comparisons test (* p < 0.01, ** p < 0.001 vs. the LDL-alone condition). All data are expressed as mean ± SEM of three independent experiments (N = 3). Each MTT assay experiment was carried out in quintuplicate.

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Western Blot, MTT Assay, Incubation, Staining, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: LDLR mRNA levels, confocal images and analysis of DiI-LDL uptake; LDLR and p-ERK expression; and cellular proliferation after siLDLR, vemurafenib (1 µM) and/or LDL (200 µg/mL ApoB) treatment compared to basal conditions for 24 h. ( A ) mRNA expression of LDLR after siLDLR for 48 h, LDL (200 µg/mL ApoB) and/or vemurafenib (1 µM) treatment in comparison to the LDL-only condition (LDL + DMSO 0.1% + Mock). ( B ) Confocal microscopy of Hoechst-stained (blue), DiI-LDL-stained (green) and lysosome-GFP (red) in BCPAP cell line after 24 h of DiI-LDL (200 µg/mL ApoB) treatment and with and without siLDLR for 48 h. The presence of colocalization of the red and green signals in the merged images is highlighted in yellow. The scale bar size was set to 50 µm. Right graph: cells were exposed to DiI-LDL (200 μg/mL ApoB) for 24 h after siLDLR for 48 h in comparison to the LDL-only condition (Mock + LDL) and analyzed for mean fluorescence intensity by fluorescence spectrometer. Statistical analysis: an unpaired t -test plus Welch’s correction were performed to compare both conditions (** p = 0.066). Data are expressed as mean ± SEM of a minimum of three independent experiments (n = 3). ( C ) Representative blot of LDLR is shown. Stain-free (SF) gel was used as the loading control. Graph shows the densitometry analysis of the Western blots of LDLR after siLDLR, LDL (200 µg/mL ApoB) incubation and/or vemurafenib treatment (1 μM) for 48 h, in comparison to the LDL-alone condition (LDL + DMSO 0.1% + Mock). ( D ) Representative blot of p-ERK is shown. Stain-free (SF) gel was used as the loading control. Graph shows the densitometry analysis of the Western blots of p-ERK after siLDLR, LDL (200 µg/mL ApoB) incubation and/or vemurafenib treatment (1 μM) for 48 h, in comparison to the LDL-only condition (LDL + DMSO 0.1% + Mock). ( E ) Proliferation percentage determined via MTT assay after incubation with LDL (200 µg/mL ApoB), vemurafenib treatment (1 µM) and/or siLDLR, compared to the LDL-only condition (LDL + DMSO 0.1% + Mock) at 48 h. Statistical analysis: one-way ANOVA test plus Tukey’s multiple comparisons test (* p < 0.01, ** p < 0.001, *** p = 0.0009, **** p < 0.0001 vs. the LDL-only condition). All data are expressed as mean ± SEM of three independent experiments (N = 3). Each MTT assay experiment was carried out in quintuplicate.

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Expressing, Comparison, Confocal Microscopy, Staining, Fluorescence, Control, Western Blot, Incubation, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells

doi: 10.3390/ijms241311153

Figure Lengend Snippet: Schematic illustration of the hypothesis that proposes a crosstalk between RAS/RAF/MAPK (MEK)/ERK pathway and LDL-mediated receptor uptake in BCPAP cell line. In the presence of LDL, a cell line harboring a BRAF mutation (BCPAP) can increase proliferation and worsens its behavior by increasing the activation of the RAS/RAF/MAPK (MEK)/ERK pathway. Vemurafenib treatment at 1uM, which enters to the cell by passive diffusion, interrupts the B-Raf/MEK step, as well as downregulating LDLR expression. Additionally, siRNA LDLR downregulates LDLR expression, decreasing LDL uptake and modulating RAS/RAF/MAPK (MEK)/ERK pathway overactivation. Black arrows mean the normal activation of the pathway, red arrows are used when the pathway is overactivated and dashed arrows symbolize when the pathway is inhibited. Part of the Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License ( https://smart.servier.com/image-set-download/ (accessed on 9 November 2022)).

Article Snippet: In terms of inhibitor treatment, the BCPAP cells were treated with 5% FBS and 0.01% DMSO as a control, with LDL (200 μg/mL ApoB) or with vemurafenib (1 μM) (PLX4032, Selleck Chemicals LLC, Houston, TX, USA) for 24 h, 48 h and 72 h. The TPC1 and BCPAP cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Minato City, Tokyo), phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA) and sodium orthovanadate (Sigma).

Techniques: Mutagenesis, Activation Assay, Diffusion-based Assay, Expressing

Journal: Cell reports

Article Title: Myeloid BAF60a deficiency alters metabolic homeostasis and exacerbates atherosclerosis

doi: 10.1016/j.celrep.2023.113171

Figure Lengend Snippet:

Article Snippet: Low-Density Lipoprotein (LDL) , Lee Biosolutions , Cat#360–10.

Techniques: Virus, Recombinant, Protease Inhibitor, Control, Saline, Magnetic Beads, Lysis, SYBR Green Assay, Transfection, EdU Assay, In Situ, Lactate Dehydrogenase Assay, Chromatin Immunoprecipitation, Gene Expression, Software, Western Blot, Modification