hot firepol evagreen qpcr supermix  (Solis BioDyne)


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    Solis BioDyne hot firepol evagreen qpcr supermix
    Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean <t>−qPCR</t> Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
    Hot Firepol Evagreen Qpcr Supermix, supplied by Solis BioDyne, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 1 article reviews
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    hot firepol evagreen qpcr supermix - by Bioz Stars, 2022-07
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    1) Product Images from "Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis"

    Article Title: Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics14010036

    Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
    Figure Legend Snippet: Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Techniques Used: Real-time Polymerase Chain Reaction

    Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
    Figure Legend Snippet: Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Techniques Used: Real-time Polymerase Chain Reaction

    2) Product Images from "Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells"

    Article Title: Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e06041

    BAPTA-AM induced sensitization by sub-toxic-dose of cisplatin is associated with a decrease in mRNA expression of p21, calmodulin, S100A8, and S100A14. A. Cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, in the presence or absence of BAPTA-AM. After treatment, total RNA was extracted from cells followed by cDNA synthesis and real-time qPCR. All mRNA expression levels were normalized against GAPDH. The data show the normalized expression of (A) p21, (B) calmodulin, (C) S100A8, and (D) S100A14, relative to the expression of the control. Each value is the mean ± SEM. Experiments were conducted with triplicate samples and repeated at least twice. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test, ∗ P
    Figure Legend Snippet: BAPTA-AM induced sensitization by sub-toxic-dose of cisplatin is associated with a decrease in mRNA expression of p21, calmodulin, S100A8, and S100A14. A. Cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, in the presence or absence of BAPTA-AM. After treatment, total RNA was extracted from cells followed by cDNA synthesis and real-time qPCR. All mRNA expression levels were normalized against GAPDH. The data show the normalized expression of (A) p21, (B) calmodulin, (C) S100A8, and (D) S100A14, relative to the expression of the control. Each value is the mean ± SEM. Experiments were conducted with triplicate samples and repeated at least twice. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test, ∗ P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Differential and dose-dependent effect of cisplatin on mRNA expression levels of p21, calmodulin, S100A8, and S100A14. After cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, mRNA expression levels were quantified by qPCR. All mRNA expression levels were normalized against GAPDH. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test. Each value is the mean ± SEM from three independent experiments, each with three replicates. ∗ P
    Figure Legend Snippet: Differential and dose-dependent effect of cisplatin on mRNA expression levels of p21, calmodulin, S100A8, and S100A14. After cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, mRNA expression levels were quantified by qPCR. All mRNA expression levels were normalized against GAPDH. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test. Each value is the mean ± SEM from three independent experiments, each with three replicates. ∗ P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    3) Product Images from "Complexome profiling on the lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins"

    Article Title: Complexome profiling on the lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins

    Journal: bioRxiv

    doi: 10.1101/2021.01.04.425283

    Growth and chlorophyll fluorescence phenotypes of the lpa2 mutant compared to wild type and complemented lines. A, Alignment of LPA2 amino acid sequences from different organisms. Predicted chloroplast transit peptides are shown in grey, predicted transmembrane helices are underlined and indicated with pipes. Peptides identified by mass spectrometry are given in red letters, twin-arginines in bold letters. Ath – Arabidopsis thaliana (AT5G51545), Zma – Zea mays (NP_001145487), Psi – Picea sitchensis (ABK23742), Ppa – Physcomitrella patens (XP_024366975), Cva – Chlorella variabilis (XP_005849843), Ota – Ostreococcus tauri (XP_003084445), Cre – Chlamydomonas reinhardtii (Cre02.g105650). B, Structure of the Chlamydomonas LPA2 gene, insertion site of the CIB1 cassette in the lpa2 mutant, and construct for complementation. Protein coding regions are drawn as black boxes, untranslated regions as bars, and introns and promoter regions as thin lines. Arrows indicate transcriptional start sites. The purple box indicates a 165-bp fragment derived from the 19th intron of gene Cre13.g573450 in reverse orientation that has integrated together with the CIB1 cassette. C, qRT-PCR analysis of LPA2 transcript accumulation. Values are means from two independent biological replicates and indicate LPA2 transcript levels in the lpa2 mutant and two complemented lines expressing the LPA2 cDNA without (c10, c11) or with a 3xHA coding region (cHA) relative to the wild type. Error bars indicate SD. D, Comparison of PSII maximum quantum efficiency (Fv/Fm). Shown are averages from 3-6 independent experiments, error bars indicate SD. Significant differences were assessed via T-test, (*** p
    Figure Legend Snippet: Growth and chlorophyll fluorescence phenotypes of the lpa2 mutant compared to wild type and complemented lines. A, Alignment of LPA2 amino acid sequences from different organisms. Predicted chloroplast transit peptides are shown in grey, predicted transmembrane helices are underlined and indicated with pipes. Peptides identified by mass spectrometry are given in red letters, twin-arginines in bold letters. Ath – Arabidopsis thaliana (AT5G51545), Zma – Zea mays (NP_001145487), Psi – Picea sitchensis (ABK23742), Ppa – Physcomitrella patens (XP_024366975), Cva – Chlorella variabilis (XP_005849843), Ota – Ostreococcus tauri (XP_003084445), Cre – Chlamydomonas reinhardtii (Cre02.g105650). B, Structure of the Chlamydomonas LPA2 gene, insertion site of the CIB1 cassette in the lpa2 mutant, and construct for complementation. Protein coding regions are drawn as black boxes, untranslated regions as bars, and introns and promoter regions as thin lines. Arrows indicate transcriptional start sites. The purple box indicates a 165-bp fragment derived from the 19th intron of gene Cre13.g573450 in reverse orientation that has integrated together with the CIB1 cassette. C, qRT-PCR analysis of LPA2 transcript accumulation. Values are means from two independent biological replicates and indicate LPA2 transcript levels in the lpa2 mutant and two complemented lines expressing the LPA2 cDNA without (c10, c11) or with a 3xHA coding region (cHA) relative to the wild type. Error bars indicate SD. D, Comparison of PSII maximum quantum efficiency (Fv/Fm). Shown are averages from 3-6 independent experiments, error bars indicate SD. Significant differences were assessed via T-test, (*** p

    Techniques Used: Fluorescence, Mutagenesis, Mass Spectrometry, Construct, Derivative Assay, Quantitative RT-PCR, Expressing

    4) Product Images from "AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats"

    Article Title: AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

    Journal: bioRxiv

    doi: 10.1101/2020.06.11.144329

    Importance of AdipoR2 to regulate membrane homeostasis. A , qPCR results showing the efficiency of different knockdowns. B , Average T half values from FRAP experiments comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 0 h PA (NT siRNA, n=7; AdipoR2 siRNA, n=6; SCD siRNA, n=5; FADS2 siRNA, n=6; ACSL4 siRNA, n=6; PEMT siRNA, n=6). C , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, siRNA after 3 h 200 μM PA (NT siRNA, n=6; AdipoR2 siRNA, n=7; SCD siRNA, n=5; FADS2 siRNA, n=5). D , E , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 6 h PA (NT siRNA, n=8; AdipoR2 siRNA, n=15; SCD siRNA, n=9; FADS2 siRNA, n=10; ACSL4 siRNA, n=8; PEMT siRNA, n=8). F , G , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 15 h PA (NT siRNA, n=5; AdipoR2 siRNA, n=13; SCD siRNA, n=11; FADS2 siRNA, n=10; ACSL4 siRNA, n=9; PEMT siRNA, n=5). H , I , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 24 h 200 μM PA c (NT siRNA, n=10; AdipoR2 siRNA, n=15; SCD siRNA, n=15; FADS2 siRNA, n=15; ACSL4 siRNA, n=15; PEMT siRNA, n=10). J , Distribution of GP index values for cells treated with NT, AdipoR2, SCD, FADS2 siRNA and challenged with PA. K-N , MUFAs and PUFAs abundance (mol%) in PCs and PEs in cells treated with different siRNAs and PA (n=4 for all treatments). O-R , PC/PE ratio, relative lysophosphatidylcholine (LPC), free cholesterol (FC) and total TAGs in cells treated with different siRNAs and PA (n=4 for all treatments). S-U , Relative gene expression for ATF4, DDiT3 and HSPA5 in cells treated different siRNAs and PA (n=4 for all treatments).
    Figure Legend Snippet: Importance of AdipoR2 to regulate membrane homeostasis. A , qPCR results showing the efficiency of different knockdowns. B , Average T half values from FRAP experiments comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 0 h PA (NT siRNA, n=7; AdipoR2 siRNA, n=6; SCD siRNA, n=5; FADS2 siRNA, n=6; ACSL4 siRNA, n=6; PEMT siRNA, n=6). C , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, siRNA after 3 h 200 μM PA (NT siRNA, n=6; AdipoR2 siRNA, n=7; SCD siRNA, n=5; FADS2 siRNA, n=5). D , E , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 6 h PA (NT siRNA, n=8; AdipoR2 siRNA, n=15; SCD siRNA, n=9; FADS2 siRNA, n=10; ACSL4 siRNA, n=8; PEMT siRNA, n=8). F , G , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 15 h PA (NT siRNA, n=5; AdipoR2 siRNA, n=13; SCD siRNA, n=11; FADS2 siRNA, n=10; ACSL4 siRNA, n=9; PEMT siRNA, n=5). H , I , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 24 h 200 μM PA c (NT siRNA, n=10; AdipoR2 siRNA, n=15; SCD siRNA, n=15; FADS2 siRNA, n=15; ACSL4 siRNA, n=15; PEMT siRNA, n=10). J , Distribution of GP index values for cells treated with NT, AdipoR2, SCD, FADS2 siRNA and challenged with PA. K-N , MUFAs and PUFAs abundance (mol%) in PCs and PEs in cells treated with different siRNAs and PA (n=4 for all treatments). O-R , PC/PE ratio, relative lysophosphatidylcholine (LPC), free cholesterol (FC) and total TAGs in cells treated with different siRNAs and PA (n=4 for all treatments). S-U , Relative gene expression for ATF4, DDiT3 and HSPA5 in cells treated different siRNAs and PA (n=4 for all treatments).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    AdipoR2 regulates membrane homeostasis to counter PA-induced lipotoxicity. A , Differential T half values comparing AdipoR2, SCD, FADS2, ACSL4 and PEMT knockdown (KD) in HEK293 cells challenged with 200 μM PA in a time-course FRAP experiment. B , Representative pseudocolor images and C , average laurdan dye GP index in HEK293 cells treated with AdipoR2, ACSL4 or FADS2 siRNA and challenged with 200 μM PA (n=15 for all treatments). D , E and F, SFAs abundance (mol%) in PCs and PEs and relative levels of TAG 48:0 in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). G , Spliced XBP1 mRNA expression in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). H , Efficiency of AdipoR2 silencing in HUVECs measured using qPCR. I , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT (nontarget) and AdipoR2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; AdipoR2 siRNA, n=3). J , AdipoR2-dependent membrane regulation to counter SFA-induced lipotoxicity: Dietary SFAs are taken up by the cells and become incorporated into membrane phospholipids (1), leading to plasma membrane rigidification (2), ER stress (3), and impairment of mitochondria function (4). In healthy cells, AdipoR2 responds to increased membrane rigidification by signalling to promote the expression of desaturases and other lipid metabolism genes (5), leading to increased levels of UFAs available for incorporation into phospholipids (6) and normalization of membrane fluidity (7). Some cells can also sequester SFAs into lipid droplets in the form of TAGs (8). A similar process occurs in C. elegans , where rigidification activates the AdipoR homolog PAQR-2 by promoting its interaction with IGLR-2.
    Figure Legend Snippet: AdipoR2 regulates membrane homeostasis to counter PA-induced lipotoxicity. A , Differential T half values comparing AdipoR2, SCD, FADS2, ACSL4 and PEMT knockdown (KD) in HEK293 cells challenged with 200 μM PA in a time-course FRAP experiment. B , Representative pseudocolor images and C , average laurdan dye GP index in HEK293 cells treated with AdipoR2, ACSL4 or FADS2 siRNA and challenged with 200 μM PA (n=15 for all treatments). D , E and F, SFAs abundance (mol%) in PCs and PEs and relative levels of TAG 48:0 in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). G , Spliced XBP1 mRNA expression in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). H , Efficiency of AdipoR2 silencing in HUVECs measured using qPCR. I , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT (nontarget) and AdipoR2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; AdipoR2 siRNA, n=3). J , AdipoR2-dependent membrane regulation to counter SFA-induced lipotoxicity: Dietary SFAs are taken up by the cells and become incorporated into membrane phospholipids (1), leading to plasma membrane rigidification (2), ER stress (3), and impairment of mitochondria function (4). In healthy cells, AdipoR2 responds to increased membrane rigidification by signalling to promote the expression of desaturases and other lipid metabolism genes (5), leading to increased levels of UFAs available for incorporation into phospholipids (6) and normalization of membrane fluidity (7). Some cells can also sequester SFAs into lipid droplets in the form of TAGs (8). A similar process occurs in C. elegans , where rigidification activates the AdipoR homolog PAQR-2 by promoting its interaction with IGLR-2.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    AdipoR2 silencing causes rigidification measured by atomic force microscopy and also alters response to different SFAs. A , qPCR results showing the efficiency of AdipoR2 knockdown in HEK293 cells; NT stands for nontarget siRNA. B , Membrane deformation measured by atomic force microscopy in HEK293 cells treated with NT and AdipoR2 siRNA under basal conditions or treated with 200 μM PA (NT siRNA and AdipoR2 siRNA Basal, n=66; NT siRNA PA, n=66; AdipoR2 siRNA PA, n=67). C , Representative deformation curve for NT and AdipoR2 siRNA-treated cells in the presence of PA D , The SFAs myristic acid (MA), PA and stearic acid (SA) all cause membrane rigidification in AdipoR2-knocked down (KD) HEK293 cells as measured using FRAP; the average T half is shown for each condition (n≥7).
    Figure Legend Snippet: AdipoR2 silencing causes rigidification measured by atomic force microscopy and also alters response to different SFAs. A , qPCR results showing the efficiency of AdipoR2 knockdown in HEK293 cells; NT stands for nontarget siRNA. B , Membrane deformation measured by atomic force microscopy in HEK293 cells treated with NT and AdipoR2 siRNA under basal conditions or treated with 200 μM PA (NT siRNA and AdipoR2 siRNA Basal, n=66; NT siRNA PA, n=66; AdipoR2 siRNA PA, n=67). C , Representative deformation curve for NT and AdipoR2 siRNA-treated cells in the presence of PA D , The SFAs myristic acid (MA), PA and stearic acid (SA) all cause membrane rigidification in AdipoR2-knocked down (KD) HEK293 cells as measured using FRAP; the average T half is shown for each condition (n≥7).

    Techniques Used: Microscopy, Real-time Polymerase Chain Reaction

    The SREBFs regulate membrane composition and fluidity in HEK293 cells challenged with 200 μM PA. A , qPCR results showing the efficiency of different knockdowns in HEK293 cells (n=3 for all treatments). B , Relative SCD expression in cells treated with different siRNAs (n=3 for all treatments). C , D Average T half values comparing cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA after C , 0 h PA challenge (n=6 for all treatments) and D , 24 h PA challenge (NT siRNA, n=10; SREBF1 siRNA, n=11; SREBF2 siRNA, n=10; SREBF1+2 siRNA, n=11). E , Distribution of GP index values for cells treated with SREBF1, SREBF2, SREBF1+2 siRNA and challenged with PA. F, Representative pseudocolor images of laurdan dye GP index and G, average GP index in HEK293 cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA and challenged with 200 μM PA (n=15 for all treatments). H-M , SFA, MUFA and PUFA abundance (mol%) in PCs and PEs in cells treated with different siRNAs and 200 μM PA (n=4 for all treatments). N , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT, SREBF1 and SREBF2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; SREBF1 siRNA, n=3; SREBF1 siRNA, n=3).
    Figure Legend Snippet: The SREBFs regulate membrane composition and fluidity in HEK293 cells challenged with 200 μM PA. A , qPCR results showing the efficiency of different knockdowns in HEK293 cells (n=3 for all treatments). B , Relative SCD expression in cells treated with different siRNAs (n=3 for all treatments). C , D Average T half values comparing cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA after C , 0 h PA challenge (n=6 for all treatments) and D , 24 h PA challenge (NT siRNA, n=10; SREBF1 siRNA, n=11; SREBF2 siRNA, n=10; SREBF1+2 siRNA, n=11). E , Distribution of GP index values for cells treated with SREBF1, SREBF2, SREBF1+2 siRNA and challenged with PA. F, Representative pseudocolor images of laurdan dye GP index and G, average GP index in HEK293 cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA and challenged with 200 μM PA (n=15 for all treatments). H-M , SFA, MUFA and PUFA abundance (mol%) in PCs and PEs in cells treated with different siRNAs and 200 μM PA (n=4 for all treatments). N , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT, SREBF1 and SREBF2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; SREBF1 siRNA, n=3; SREBF1 siRNA, n=3).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    5) Product Images from "Nicotinic Antagonist UFR2709 Inhibits Nicotine Reward and Decreases Anxiety in Zebrafish"

    Article Title: Nicotinic Antagonist UFR2709 Inhibits Nicotine Reward and Decreases Anxiety in Zebrafish

    Journal: Molecules

    doi: 10.3390/molecules25132998

    ( A ) Gel electrophoresis of PCR products in control (untreated) animals. Lanes 1: 100 bp DNA ladder; 2: No-RT control; 3: β-Actin; 4: nAChR subunit α4; 5: nAChR subunit α7. ( B ) Expression change of nAChR subunits α4 and α7 after nicotine or UFR2709 treatments (qPCR). * p
    Figure Legend Snippet: ( A ) Gel electrophoresis of PCR products in control (untreated) animals. Lanes 1: 100 bp DNA ladder; 2: No-RT control; 3: β-Actin; 4: nAChR subunit α4; 5: nAChR subunit α7. ( B ) Expression change of nAChR subunits α4 and α7 after nicotine or UFR2709 treatments (qPCR). * p

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    6) Product Images from "Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins"

    Article Title: Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008975

    Overexpression of AdipoR1 or AdipoR2 compensates for the absence of AdipoR2 in mammalian cells. (A) Western blot detection of VSV-AdipoR1-FLAG transiently expressed in HEK293 cells, with GAPDH used as a loading control. (B) Snapshots from a typical FRAP experiment, with the bleached areas (large circle) and reference area (small circle) indicated before and after bleaching. (C) T half from FRAP experiments showing that the addition of 400 μM palmitic acid causes membrane rigidification and that this effect is attenuated by AdipoR1 overexpression (R1OE). (D) Western blot detection of HA-AdipoR2-MYC transiently expressed in HEK293 cells, with GAPDH used as a loading control. (E) T half from FRAP experiments showing that the addition of 400 μM palmitic acid causes membrane rigidification and that this effect is attenuated by AdipoR2 overexpression (R2OE). (F) Efficiency of the AdipoR2 siRNA knockdown normalized to non-target siRNA (NT siRNA) and quantified using QPCR. ( G) T half from FRAP experiments showing that the addition of overexpression of AdipoR1 (R1OE) prevents membrane rigidification in cells where AdipoR2 has been silenced by siRNA. (H) FRAP curves for the AdipoR2 siRNA-treated samples from G; note the improved fluorescence recovery rate when AdipoR1 is overexpressed (R1OE). Significant differences are indicated where: * p
    Figure Legend Snippet: Overexpression of AdipoR1 or AdipoR2 compensates for the absence of AdipoR2 in mammalian cells. (A) Western blot detection of VSV-AdipoR1-FLAG transiently expressed in HEK293 cells, with GAPDH used as a loading control. (B) Snapshots from a typical FRAP experiment, with the bleached areas (large circle) and reference area (small circle) indicated before and after bleaching. (C) T half from FRAP experiments showing that the addition of 400 μM palmitic acid causes membrane rigidification and that this effect is attenuated by AdipoR1 overexpression (R1OE). (D) Western blot detection of HA-AdipoR2-MYC transiently expressed in HEK293 cells, with GAPDH used as a loading control. (E) T half from FRAP experiments showing that the addition of 400 μM palmitic acid causes membrane rigidification and that this effect is attenuated by AdipoR2 overexpression (R2OE). (F) Efficiency of the AdipoR2 siRNA knockdown normalized to non-target siRNA (NT siRNA) and quantified using QPCR. ( G) T half from FRAP experiments showing that the addition of overexpression of AdipoR1 (R1OE) prevents membrane rigidification in cells where AdipoR2 has been silenced by siRNA. (H) FRAP curves for the AdipoR2 siRNA-treated samples from G; note the improved fluorescence recovery rate when AdipoR1 is overexpressed (R1OE). Significant differences are indicated where: * p

    Techniques Used: Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Fluorescence

    7) Product Images from "Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans"

    Article Title: Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans

    Journal: Current Genetics

    doi: 10.1007/s00294-016-0644-9

    Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of ( a ) atf21 + , ( b ) gpd1 + , and ( c ) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 ( mak1Δ ), PJ1643 ( mak1,2Δ ), PJ1644 ( mak1,3Δ ), PJ1645 ( mak2,3Δ ), and PJ1646 ( mak1,2,3Δ ). The levels of mRNA transcripts were measured under non-induced conditions ( black bars ) and upon 15 min of treatment with 1 M sorbitol ( grey bars ). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1 + mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003 )
    Figure Legend Snippet: Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of ( a ) atf21 + , ( b ) gpd1 + , and ( c ) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 ( mak1Δ ), PJ1643 ( mak1,2Δ ), PJ1644 ( mak1,3Δ ), PJ1645 ( mak2,3Δ ), and PJ1646 ( mak1,2,3Δ ). The levels of mRNA transcripts were measured under non-induced conditions ( black bars ) and upon 15 min of treatment with 1 M sorbitol ( grey bars ). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1 + mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003 )

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    8) Product Images from "Vitamin C Fosters the In Vivo Differentiation of Peripheral CD4+ Foxp3− T Cells into CD4+ Foxp3+ Regulatory T Cells but Impairs Their Ability to Prolong Skin Allograft Survival"

    Article Title: Vitamin C Fosters the In Vivo Differentiation of Peripheral CD4+ Foxp3− T Cells into CD4+ Foxp3+ Regulatory T Cells but Impairs Their Ability to Prolong Skin Allograft Survival

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00112

    CD4 +  Foxp3/GFP +  regulatory T cells express the highest levels of SVCT2 expression among other leukocyte populations. Antigen presenting cells (APCs), dendritic cells, B cells, CD8 +  T cells, total CD4 +  T cells, CD4 +  Foxp3/GFP +  [regulatory T cell (Treg)], and CD4 +  Foxp3/GFP −  T cells (Tconv) were isolated from the spleens of naive Foxp3/GFP reporter mice.  (A)  Real-time PCR (RT-PCR) for SVCT2 and housekeeping gene 18S in isolated immune cells.  (B,C)  Quantification of SVCT2 expression relative to 18S, by RT-PCR in freshly isolated immune cells  (B) , and sorted Tregs and Tconv after 3 days of polyclonal activation  (C) . Data represent mean ± SD ( n  = 5 of two independent experiments in B and  n  = 3 of three independent experiments in C). Parametric statistical analysis with unpaired Student’s  t -test was performed. * P
    Figure Legend Snippet: CD4 + Foxp3/GFP + regulatory T cells express the highest levels of SVCT2 expression among other leukocyte populations. Antigen presenting cells (APCs), dendritic cells, B cells, CD8 + T cells, total CD4 + T cells, CD4 + Foxp3/GFP + [regulatory T cell (Treg)], and CD4 + Foxp3/GFP − T cells (Tconv) were isolated from the spleens of naive Foxp3/GFP reporter mice. (A) Real-time PCR (RT-PCR) for SVCT2 and housekeeping gene 18S in isolated immune cells. (B,C) Quantification of SVCT2 expression relative to 18S, by RT-PCR in freshly isolated immune cells (B) , and sorted Tregs and Tconv after 3 days of polyclonal activation (C) . Data represent mean ± SD ( n  = 5 of two independent experiments in B and n  = 3 of three independent experiments in C). Parametric statistical analysis with unpaired Student’s t -test was performed. * P

    Techniques Used: Expressing, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Activation Assay

    9) Product Images from "Mitochondrial STAT3 regulates proliferation of tissue stem cells"

    Article Title: Mitochondrial STAT3 regulates proliferation of tissue stem cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.17.208264

    Validation of the MLS_Stat3_NES construct in murine Embryonic Stem Cells. A: Western blot for total STAT3 on Stat3 +/+ , stat3 −/− and MLS_Stat3_NES cells. Note the shift in molecular weight due to the presence of MLS and NES tags. STAT3 protein level in both MLS_Stat3_NES clones is lower than Stat3 +/+ cells. B: qPCR analysis of the Stat3 and its nuclear target gene Socs3 . Gene expression analysis of Stat3 +/+ cells, stat3 −/− cells, and two MLS_Stat3_NES clones (A/B) cultured in presence of LIF. Note that both clones have the same undetectable level of Socs3 as stat3 −/− cells. C: Representative confocal images of Stat3 +/+ , stat3 −/− and MLS_Stat3_NES cells stained with anti-STAT3 and anti-ATAD3 antibodies. Merge image shows co-localization between STAT3 and the nucleoids marked by ATAD3; DAPI serves as a nuclear counterstain. Scale bar: 20 μm.
    Figure Legend Snippet: Validation of the MLS_Stat3_NES construct in murine Embryonic Stem Cells. A: Western blot for total STAT3 on Stat3 +/+ , stat3 −/− and MLS_Stat3_NES cells. Note the shift in molecular weight due to the presence of MLS and NES tags. STAT3 protein level in both MLS_Stat3_NES clones is lower than Stat3 +/+ cells. B: qPCR analysis of the Stat3 and its nuclear target gene Socs3 . Gene expression analysis of Stat3 +/+ cells, stat3 −/− cells, and two MLS_Stat3_NES clones (A/B) cultured in presence of LIF. Note that both clones have the same undetectable level of Socs3 as stat3 −/− cells. C: Representative confocal images of Stat3 +/+ , stat3 −/− and MLS_Stat3_NES cells stained with anti-STAT3 and anti-ATAD3 antibodies. Merge image shows co-localization between STAT3 and the nucleoids marked by ATAD3; DAPI serves as a nuclear counterstain. Scale bar: 20 μm.

    Techniques Used: Construct, Western Blot, Molecular Weight, Clone Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Staining

    MEK-ERK pathway regulates mitoStat3-driven cell proliferation by phosphorylating STAT3 on S727 residue. A: qRT-PCR analysis of mt_nd2 and pcna in 48-hpf larvae treated with 12.5 μM PD98059 from 24-48 hpf. B: representative pictures of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. B’: fluorescence quantification of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. B’’: number of EGFP positive cells in intestines of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. Statistical analysis was performed by unpaired t-test on 3 independent biological samples. *p
    Figure Legend Snippet: MEK-ERK pathway regulates mitoStat3-driven cell proliferation by phosphorylating STAT3 on S727 residue. A: qRT-PCR analysis of mt_nd2 and pcna in 48-hpf larvae treated with 12.5 μM PD98059 from 24-48 hpf. B: representative pictures of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. B’: fluorescence quantification of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. B’’: number of EGFP positive cells in intestines of Tg(7xStat3:EGFP) transgenic larvae treated with 12.5 μM PD98059 from 24 dpf to 96 dpf. Statistical analysis was performed by unpaired t-test on 3 independent biological samples. *p

    Techniques Used: Quantitative RT-PCR, Transgenic Assay, Fluorescence

    10) Product Images from "Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2"

    Article Title: Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2

    Journal: eLife

    doi: 10.7554/eLife.40686

    Protective effects of TLCD1/2 knockdown against AdipoR2 knockdown. ( A ) qPCR quantification of the siRNA knockdown efficiency against AdipoR2 and TLCD2. ( B–E ) Effect of AdipoR2, TLCD1, TLCD2 knockdown and 5 μM EPA on ceramide composition. Note that exogenous TLCD1 and TLCD2 knockdown have effects similar to that of exogenous EPA. 10.7554/eLife.40686.039 Lipidomics data used in panels B-E
    Figure Legend Snippet: Protective effects of TLCD1/2 knockdown against AdipoR2 knockdown. ( A ) qPCR quantification of the siRNA knockdown efficiency against AdipoR2 and TLCD2. ( B–E ) Effect of AdipoR2, TLCD1, TLCD2 knockdown and 5 μM EPA on ceramide composition. Note that exogenous TLCD1 and TLCD2 knockdown have effects similar to that of exogenous EPA. 10.7554/eLife.40686.039 Lipidomics data used in panels B-E

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of TLCD1/2 knockdown on FA composition and membrane fluidity. siRNA knockdown does not cause large changes in the FA composition of PCs ( A ), PEs ( B ), PC/PE ratio ( C ), free cholesterol-to-PC ratio ( D ), or ceramide levels, which are strongly affected by cultivation in the presence of PA ( E–H ). TLCD2 siRNA does not cause any increase in TAG stores or in EPA storage in TAGs ( I–J ). ( K–L ) Amount of LA-[ 13 C 18 ] present in PCs and PEs at 6, 12 and 24 hr after its removals; the cells were treated with the indicated siRNA (NT, TLCD1 or TLCD2) and pre-incubated for 24 hr in the presence of LA-[ 13 C 18 ] prior to its removal. ( M ) qPCR quantification of the siRNA knockdown efficiency against FADS2. Symbols for statistical significance: * TLCD1 or TLCD2 > NT; # NT > TLCD1; $ NT > TLCD2; in panel K ), NT differed from TLCD1 and TLCD2 with p
    Figure Legend Snippet: Effect of TLCD1/2 knockdown on FA composition and membrane fluidity. siRNA knockdown does not cause large changes in the FA composition of PCs ( A ), PEs ( B ), PC/PE ratio ( C ), free cholesterol-to-PC ratio ( D ), or ceramide levels, which are strongly affected by cultivation in the presence of PA ( E–H ). TLCD2 siRNA does not cause any increase in TAG stores or in EPA storage in TAGs ( I–J ). ( K–L ) Amount of LA-[ 13 C 18 ] present in PCs and PEs at 6, 12 and 24 hr after its removals; the cells were treated with the indicated siRNA (NT, TLCD1 or TLCD2) and pre-incubated for 24 hr in the presence of LA-[ 13 C 18 ] prior to its removal. ( M ) qPCR quantification of the siRNA knockdown efficiency against FADS2. Symbols for statistical significance: * TLCD1 or TLCD2 > NT; # NT > TLCD1; $ NT > TLCD2; in panel K ), NT differed from TLCD1 and TLCD2 with p

    Techniques Used: Incubation, Real-time Polymerase Chain Reaction

    Specificity of the TLCD1/2 siRNA effects. ( A ) QPCR quantification of TLCD1 and TLCD2 48 or 72 hr after siRNA treatment and normalized to cells treated with NT siRNA. ( B ) Image of BODIPY 500/510 C 1 , C 12 -labelled HEK293 cells. Note the widespread fluorescence throughout what appears to be most or all cellular membranes. ( C–D ) TLCD1 and TLCD2 siRNA have no effect on the membrane fluidity of HEK293 cells grown in normal media. ( E ) 400 μM PA causes a decrease in the membrane fluidity of HEK293 cells treated with control NT siRNA. ( F–G ) siRNA against the control gene PPIB does not protect against the rigidifying effects of PA. ( H ) TLCD1 or TLCD2 knockdown protect against PA-induced apoptosis; apoptotic cells are in the lower right quadrant of each panel. ( I ) Summary of several apoptosis quantification assays. Note that TLCD1 and TLCD2 knockdown is beneficial to HEK293 cells both in basal conditions and when challenged with PA. ( J ) The uptake rate of tritiated PA is not affected by TLCD1 or TLCD2 knockdown; phloretin was used as a positive control and strongly inhibited the uptake. ( K–L ) PA treatment causes an increase in the SFA content in TAGs and also raises the TAG/PC ratio; neither of these PA effects is inhibited by TLCD1 or TLCD2 siRNA. ( M–N ) Neither TLCD1 nor TLCD2 siRNA had an effect on Nile red staining of lipid stores in HEK2393 cultivated with or without PA, suggesting that these genes do not affect FA sequestration. ( O ) siRNA against TLCD1 or TLCD2 did not cause an increase in the expression of four tested desaturases; indeed, they significantly caused a decrease in the expression of SCD. 10.7554/eLife.40686.025 Lipidomics data for panels K-L.
    Figure Legend Snippet: Specificity of the TLCD1/2 siRNA effects. ( A ) QPCR quantification of TLCD1 and TLCD2 48 or 72 hr after siRNA treatment and normalized to cells treated with NT siRNA. ( B ) Image of BODIPY 500/510 C 1 , C 12 -labelled HEK293 cells. Note the widespread fluorescence throughout what appears to be most or all cellular membranes. ( C–D ) TLCD1 and TLCD2 siRNA have no effect on the membrane fluidity of HEK293 cells grown in normal media. ( E ) 400 μM PA causes a decrease in the membrane fluidity of HEK293 cells treated with control NT siRNA. ( F–G ) siRNA against the control gene PPIB does not protect against the rigidifying effects of PA. ( H ) TLCD1 or TLCD2 knockdown protect against PA-induced apoptosis; apoptotic cells are in the lower right quadrant of each panel. ( I ) Summary of several apoptosis quantification assays. Note that TLCD1 and TLCD2 knockdown is beneficial to HEK293 cells both in basal conditions and when challenged with PA. ( J ) The uptake rate of tritiated PA is not affected by TLCD1 or TLCD2 knockdown; phloretin was used as a positive control and strongly inhibited the uptake. ( K–L ) PA treatment causes an increase in the SFA content in TAGs and also raises the TAG/PC ratio; neither of these PA effects is inhibited by TLCD1 or TLCD2 siRNA. ( M–N ) Neither TLCD1 nor TLCD2 siRNA had an effect on Nile red staining of lipid stores in HEK2393 cultivated with or without PA, suggesting that these genes do not affect FA sequestration. ( O ) siRNA against TLCD1 or TLCD2 did not cause an increase in the expression of four tested desaturases; indeed, they significantly caused a decrease in the expression of SCD. 10.7554/eLife.40686.025 Lipidomics data for panels K-L.

    Techniques Used: Real-time Polymerase Chain Reaction, Fluorescence, Positive Control, Staining, Expressing

    11) Product Images from "The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids"

    Article Title: The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007004

    Mammalian AdipoR2 prevents rigidification by palmitic acid. (A) FRAP analysis showing that 400 μM PA causes reduced membrane fluidity in HEK293 cells and that 400 μM OA causes increased membrane fluidity even when PA is included. (B) T half values from FRAP analyses performed with different concentrations of PA and OA. (C) Quantitative PCR results showing the degree of siRNA inhibition of the indicated genes. (D-G) The morphology of HEK293 cells is altered by PA when AdipoR2 is knocked down. Note the presence of numerous circular structures in the BODIPY-labeled cells treated with AdipoR2 siRNA (arrows). Nuclei are indicated by the letter
    Figure Legend Snippet: Mammalian AdipoR2 prevents rigidification by palmitic acid. (A) FRAP analysis showing that 400 μM PA causes reduced membrane fluidity in HEK293 cells and that 400 μM OA causes increased membrane fluidity even when PA is included. (B) T half values from FRAP analyses performed with different concentrations of PA and OA. (C) Quantitative PCR results showing the degree of siRNA inhibition of the indicated genes. (D-G) The morphology of HEK293 cells is altered by PA when AdipoR2 is knocked down. Note the presence of numerous circular structures in the BODIPY-labeled cells treated with AdipoR2 siRNA (arrows). Nuclei are indicated by the letter "N", and the circle in (D) indicates the size of the area bleached in FRAP experiments with HEK293 cells. (H-K) FRAP analyses showing that AdipoR2 knockdown greatly increases the rigidifying effect of PA. (L-O) Lipidomics analysis of HEK 293 cells cultivated 24 hours in serum free media or serum-free media containing PA, OA or PA + OA and treated with non-target siRNA (NT) or AdipoR1 or AdipoR2 siRNA, as indicated. Note that PA alone causes a dramatic increase in SFAs among PCs, PEs and TAGs, and that this effect is increased by AdipoR2 knockdown. (P-Q ) FRAP analyses showing that AdipoR2 knockdown greatly increases the rigidifying effect of 200 μM PA. (R-S) Lipidomics analysis showing that AdipoR2 siRNA also causes an excess of SFAs among PCs and PEs when the cells are incubated with 200 μM PA. Statistical analysis in L-O and R-S were done by comparing siRNA-treated cells with the non-target siRNA cultivated under the same conditions.

    Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Labeling, Incubation

    12) Product Images from "The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria"

    Article Title: The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.037226

    Biochemical and genetic characterization of mitochondrial phenotype of  mpv17  KO larvae.  (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and  mpv17 −/−  mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and  mpv17  homozygous mutants. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P
    Figure Legend Snippet: Biochemical and genetic characterization of mitochondrial phenotype of mpv17 KO larvae. (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and mpv17 −/− mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and mpv17 homozygous mutants. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Techniques Used: Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Two Tailed Test

    Evaluation of stress response and mitochondrial quality control system in zebrafish larvae.  (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P
    Figure Legend Snippet: Evaluation of stress response and mitochondrial quality control system in zebrafish larvae. (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    Investigation of  mpv17  orthologue and paralogue genes in zebrafish larvae.  (A) Real-time PCR quantification of mRNA transcripts of  mpv17-like2  and  mpv17-like  at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human  MPV17  mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish  mpv17  and  mpv17-like2  mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in  mpv17 −/−  mutants transiently overexpressing  mpv17-like2  at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P
    Figure Legend Snippet: Investigation of mpv17 orthologue and paralogue genes in zebrafish larvae. (A) Real-time PCR quantification of mRNA transcripts of mpv17-like2 and mpv17-like at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human MPV17 mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish mpv17 and mpv17-like2 mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in mpv17 −/− mutants transiently overexpressing mpv17-like2 at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Techniques Used: Real-time Polymerase Chain Reaction, Injection, Two Tailed Test

    13) Product Images from "Possible Role of Circulating Bone Marrow Mesenchymal Progenitors in Modulating Inflammation and Promoting Wound Repair"

    Article Title: Possible Role of Circulating Bone Marrow Mesenchymal Progenitors in Modulating Inflammation and Promoting Wound Repair

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23010078

    mRNA expression of genes involved in mesenchymal and haematopoietic lineages was determined by qPCR. Transcript levels were normalized to the ACTB reference gene using log2 (2-ΔCt) method. The data is presented as mean ± standard error (SE). The graph bar shows expression level of the genes CD105, CD29, CD45, and CD34.
    Figure Legend Snippet: mRNA expression of genes involved in mesenchymal and haematopoietic lineages was determined by qPCR. Transcript levels were normalized to the ACTB reference gene using log2 (2-ΔCt) method. The data is presented as mean ± standard error (SE). The graph bar shows expression level of the genes CD105, CD29, CD45, and CD34.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    ( A ) Experimental wound model. Schematic representation of the nine experimental conditions: unscratched skin (control, TNFα, and HGF supplemented), scratched skin (scratched, TNFα, and HGF supplemented), and scratched skin and BMMPs infused (scratched, TNFα, and HGF supplemented, followed by BMMPs infusion). The scheme also shows the three time points taken into consideration (days one, three, and five after wound infliction). qPCR and ELISA were performed to measure expression levels of IL-8 and CD73 and protein levels of IL-8, SCF, and FGF-1, respectively ( B ). Schematic representation of the different experimental conditions on skin models on day three after wound infliction ( C ). The schemes show the effect of TNFα ( A ) and HGF ( B ) supplementation and BMMPs infusion with and in absence of damage. Evaluation of their effects was conducted at molecular level, by gene expression level quantification of IL-8 and CD73 , and at protein level measurement of IL-8 and SCF. Created with BioRender.com. https://biorender.com/ (Accessed on 15 November 2021).
    Figure Legend Snippet: ( A ) Experimental wound model. Schematic representation of the nine experimental conditions: unscratched skin (control, TNFα, and HGF supplemented), scratched skin (scratched, TNFα, and HGF supplemented), and scratched skin and BMMPs infused (scratched, TNFα, and HGF supplemented, followed by BMMPs infusion). The scheme also shows the three time points taken into consideration (days one, three, and five after wound infliction). qPCR and ELISA were performed to measure expression levels of IL-8 and CD73 and protein levels of IL-8, SCF, and FGF-1, respectively ( B ). Schematic representation of the different experimental conditions on skin models on day three after wound infliction ( C ). The schemes show the effect of TNFα ( A ) and HGF ( B ) supplementation and BMMPs infusion with and in absence of damage. Evaluation of their effects was conducted at molecular level, by gene expression level quantification of IL-8 and CD73 , and at protein level measurement of IL-8 and SCF. Created with BioRender.com. https://biorender.com/ (Accessed on 15 November 2021).

    Techniques Used: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing

    14) Product Images from "Membrane Fluidity Is Regulated Cell Nonautonomously by Caenorhabditis elegans PAQR-2 and Its Mammalian Homolog AdipoR2"

    Article Title: Membrane Fluidity Is Regulated Cell Nonautonomously by Caenorhabditis elegans PAQR-2 and Its Mammalian Homolog AdipoR2

    Journal: Genetics

    doi: 10.1534/genetics.118.301272

    AdipoR2-positive HEK293 cells contribute to membrane homeostasis in distant cells. (A) Photograph of the center of a partitioned culture dish, after removal of the partition. Note the HEK293 cells growing in four-well separated quadrants. (B) Quantitative PCR showing that AdipoR2 siRNA lowers AdipoR2 messenger RNA levels to
    Figure Legend Snippet: AdipoR2-positive HEK293 cells contribute to membrane homeostasis in distant cells. (A) Photograph of the center of a partitioned culture dish, after removal of the partition. Note the HEK293 cells growing in four-well separated quadrants. (B) Quantitative PCR showing that AdipoR2 siRNA lowers AdipoR2 messenger RNA levels to

    Techniques Used: Real-time Polymerase Chain Reaction

    15) Product Images from "SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells"

    Article Title: SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2016.12.001

    Loss of SNEV and WRN Reduces Fat Deposition in C. elegans (A) Verification of RNAi efficacy upon depletion of prp-19 , wrn-1 , xpa-1 , and M18.5 in wild-type C. elegans by qPCR. Knockdown efficiency was always between 80% and 100%. Error bars represent SEM of four technical replicates. (B and C) Oil red O staining demonstrates significantly lower fat deposition upon prp-19 knockdown, compared with the empty vector (HT115) control. Representative images (B) and quantification (C) of three biological replicates with at least 20 worms per strain are shown. Error bars represent SEM. ∗∗∗ p
    Figure Legend Snippet: Loss of SNEV and WRN Reduces Fat Deposition in C. elegans (A) Verification of RNAi efficacy upon depletion of prp-19 , wrn-1 , xpa-1 , and M18.5 in wild-type C. elegans by qPCR. Knockdown efficiency was always between 80% and 100%. Error bars represent SEM of four technical replicates. (B and C) Oil red O staining demonstrates significantly lower fat deposition upon prp-19 knockdown, compared with the empty vector (HT115) control. Representative images (B) and quantification (C) of three biological replicates with at least 20 worms per strain are shown. Error bars represent SEM. ∗∗∗ p

    Techniques Used: Real-time Polymerase Chain Reaction, Staining, Plasmid Preparation

    16) Product Images from "NF-kB pathway is involved in microscopic colitis pathogenesis"

    Article Title: NF-kB pathway is involved in microscopic colitis pathogenesis

    Journal: The Journal of International Medical Research

    doi: 10.1177/03000605221080104

    Real-time polymerase chain reaction (qRT-PCR) gene expression results for formalin-fixed and paraffin-embedded samples from a different group of patients with microscopic colitis (MC) compared with health control (HC) tissues undertaken to verify the results for the four genes identified by the RT 2  Profiler PCR Array. Data presented as mean ± SD; * P  ≤ 0.05; Student’s  t -test. CCL19, C-C motif chemokine ligand 19; CCL21, C-C motif chemokine ligand 21; C3, complement C3; LTB, lymphotoxin beta.
    Figure Legend Snippet: Real-time polymerase chain reaction (qRT-PCR) gene expression results for formalin-fixed and paraffin-embedded samples from a different group of patients with microscopic colitis (MC) compared with health control (HC) tissues undertaken to verify the results for the four genes identified by the RT 2 Profiler PCR Array. Data presented as mean ± SD; * P  ≤ 0.05; Student’s t -test. CCL19, C-C motif chemokine ligand 19; CCL21, C-C motif chemokine ligand 21; C3, complement C3; LTB, lymphotoxin beta.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Polymerase Chain Reaction

    17) Product Images from "dsRNA Molecules From the Tobacco Mosaic Virus p126 Gene Counteract TMV-Induced Proteome Changes at an Early Stage of Infection"

    Article Title: dsRNA Molecules From the Tobacco Mosaic Virus p126 Gene Counteract TMV-Induced Proteome Changes at an Early Stage of Infection

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2021.663707

    Analysis of TMV p126 expression levels in TMV and dsRNAp126 + TMV treatments by RT-PCR.  (A)  Gel images from two biological replicates (Rep 1 and Rep 2) showing p126 RNA abundance by semiquantitative RT-PCR. RNA samples were collected at two time points (15 min and 24 h post treatment). Primers for the detection of p126 were designed outside the region (namely 426–1,091 of the TMV genome) that was used for the production of dsRNAp126. Nt-ACT9 was employed as an endogenous reference gene. H 2 O indicates samples collected from the negative control treatment. M is a low molecular weight DNA marker (New England Biolabs, United States).  (B)  Relative quantification of p126 expression levels by RT quantitative PCR, employing Nt-ACT9 for normalization purposes. Depicted are the log 2 -transformed values of p126 abundance in TMV (light-gray columns) and dsRNAp126 + TMV (dark-gray columns) treatments. The value of p126 at 15 min in TMV treatment was set as 1 (log 2  value was 0). Bars represent standard errors. Statistical analysis was performed per time point using Student’s  t -test. Asterisks indicate significant differences between TMV and dsRNAp126 + TMV treatments at the respective time points (* p
    Figure Legend Snippet: Analysis of TMV p126 expression levels in TMV and dsRNAp126 + TMV treatments by RT-PCR. (A) Gel images from two biological replicates (Rep 1 and Rep 2) showing p126 RNA abundance by semiquantitative RT-PCR. RNA samples were collected at two time points (15 min and 24 h post treatment). Primers for the detection of p126 were designed outside the region (namely 426–1,091 of the TMV genome) that was used for the production of dsRNAp126. Nt-ACT9 was employed as an endogenous reference gene. H 2 O indicates samples collected from the negative control treatment. M is a low molecular weight DNA marker (New England Biolabs, United States). (B) Relative quantification of p126 expression levels by RT quantitative PCR, employing Nt-ACT9 for normalization purposes. Depicted are the log 2 -transformed values of p126 abundance in TMV (light-gray columns) and dsRNAp126 + TMV (dark-gray columns) treatments. The value of p126 at 15 min in TMV treatment was set as 1 (log 2 value was 0). Bars represent standard errors. Statistical analysis was performed per time point using Student’s t -test. Asterisks indicate significant differences between TMV and dsRNAp126 + TMV treatments at the respective time points (* p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Molecular Weight, Marker, Real-time Polymerase Chain Reaction, Transformation Assay

    18) Product Images from "Bioaugmented sand filter columns provide stable removal of pesticide residue from membrane retentate"

    Article Title: Bioaugmented sand filter columns provide stable removal of pesticide residue from membrane retentate

    Journal: bioRxiv

    doi: 10.1101/2020.06.04.135582

    Number of Aminobacter sp. MSH1 cells (determined by qPCR applying the newly designed specific primers) detected in the sand sampled from different locations within the columns at experimental termination. Columns received during operation a feed of either untreated water with 0.3 µg l -1 BAM (light blue) or membrane retentate water with 3.0 µg l -1 BAM (dark blue bars). For the columns inoculated with Aminobacter sp. MSH1 data are presented as means (n = 3) with error bars showing standard error. Two control columns were run without initial inoculation (hatched bars; n = 1).
    Figure Legend Snippet: Number of Aminobacter sp. MSH1 cells (determined by qPCR applying the newly designed specific primers) detected in the sand sampled from different locations within the columns at experimental termination. Columns received during operation a feed of either untreated water with 0.3 µg l -1 BAM (light blue) or membrane retentate water with 3.0 µg l -1 BAM (dark blue bars). For the columns inoculated with Aminobacter sp. MSH1 data are presented as means (n = 3) with error bars showing standard error. Two control columns were run without initial inoculation (hatched bars; n = 1).

    Techniques Used: Real-time Polymerase Chain Reaction

    19) Product Images from "Y705 and S727 are required for mitochondrial import and transcriptional activities of STAT3 and regulate proliferation of embryonic and tissue stem cells"

    Article Title: Y705 and S727 are required for mitochondrial import and transcriptional activities of STAT3 and regulate proliferation of embryonic and tissue stem cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.17.208264

    Validation of the MLS_Stat3_NES construct in murine Embryonic Stem Cells. A: Western blot for total STAT3 on Stat3 +/+ , Stat3 -/- and MLS_Stat3_NES cells. Note the shift in molecular weight due to the presence of MLS and NES tags. STAT3 protein level in both MLS_Stat3_NES clones is lower than Stat3 +/+ cells. B: qPCR analysis of the Stat3 and its nuclear target gene Socs3. Gene expression analysis of Stat3 +/+ cells, Stat3 -/- cells, and two MLS_Stat3_NES clones (A/B) cultured in presence of LIF. Note that both clones have the same undetectable level of Socs3 as Stat3 -/- cells. C: Representative confocal images of Stat3 +/+ , Stat3 -/- and MLS_Stat3_NES cells stained with anti-STAT3 and anti-ATAD3 antibodies. Merge image shows co-localization between STAT3 and the nucleoids marked by ATAD3; DAPI serves as a nuclear counterstain. Scale bar: 20 μm.
    Figure Legend Snippet: Validation of the MLS_Stat3_NES construct in murine Embryonic Stem Cells. A: Western blot for total STAT3 on Stat3 +/+ , Stat3 -/- and MLS_Stat3_NES cells. Note the shift in molecular weight due to the presence of MLS and NES tags. STAT3 protein level in both MLS_Stat3_NES clones is lower than Stat3 +/+ cells. B: qPCR analysis of the Stat3 and its nuclear target gene Socs3. Gene expression analysis of Stat3 +/+ cells, Stat3 -/- cells, and two MLS_Stat3_NES clones (A/B) cultured in presence of LIF. Note that both clones have the same undetectable level of Socs3 as Stat3 -/- cells. C: Representative confocal images of Stat3 +/+ , Stat3 -/- and MLS_Stat3_NES cells stained with anti-STAT3 and anti-ATAD3 antibodies. Merge image shows co-localization between STAT3 and the nucleoids marked by ATAD3; DAPI serves as a nuclear counterstain. Scale bar: 20 μm.

    Techniques Used: Construct, Western Blot, Molecular Weight, Clone Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Staining

    20) Product Images from "Bioaugmented sand filter columns provide stable removal of pesticide residue from membrane retentate"

    Article Title: Bioaugmented sand filter columns provide stable removal of pesticide residue from membrane retentate

    Journal: bioRxiv

    doi: 10.1101/2020.06.04.135582

    Number of Aminobacter sp. MSH1 cells (determined by qPCR applying the newly designed specific primers) detected in the sand sampled from different locations within the columns at experimental termination. Columns received during operation a feed of either untreated water with 0.3 µg l -1 BAM (light blue) or membrane retentate water with 3.0 µg l -1 BAM (dark blue bars). For the columns inoculated with Aminobacter sp. MSH1 data are presented as means (n = 3) with error bars showing standard error. Two control columns were run without initial inoculation (hatched bars; n = 1).
    Figure Legend Snippet: Number of Aminobacter sp. MSH1 cells (determined by qPCR applying the newly designed specific primers) detected in the sand sampled from different locations within the columns at experimental termination. Columns received during operation a feed of either untreated water with 0.3 µg l -1 BAM (light blue) or membrane retentate water with 3.0 µg l -1 BAM (dark blue bars). For the columns inoculated with Aminobacter sp. MSH1 data are presented as means (n = 3) with error bars showing standard error. Two control columns were run without initial inoculation (hatched bars; n = 1).

    Techniques Used: Real-time Polymerase Chain Reaction

    21) Product Images from "Hepatogenic Potential and Liver Regeneration Effect of Human Liver-derived Mesenchymal-Like Stem Cells"

    Article Title: Hepatogenic Potential and Liver Regeneration Effect of Human Liver-derived Mesenchymal-Like Stem Cells

    Journal: Cells

    doi: 10.3390/cells9061521

    Comparison of the characteristics between human liver-derived stem cells (hLD-SCs) and mesenchymal stromal/stem cells (MSCs) from other origins. ( A ) Microscopy imaging of human umbilical cord matrix derived stem cell (hUC-MSCs), human bone marrow stem cell (hBM-MSCs), and human liver-derived stem cells (hLD-SCs) isolated from three different individuals. Scale bar, 200 μm. ( B ) Flow cytometry measurement of hUC-MSCs, hBM-MSCs, and three hLD-SCs. CD34 was labeled as a hematopoietic stem cell marker. CD90 and CD105 were labeled as mesenchymal stem cell markers. ( C – E ) Quantitative real-time PCR (qRT-PCR) analysis of ( C ) pluripotency markers ( OCT4 ,  NANOG , and  SOX2 ), ( D ) pro-inflammatory cytokines ( TNFα ,  IL1β,  and  IL6 ), and ( E ) anti-inflammatory cytokines ( TGFβ1  and  IL10 ). Gene expression levels were normalized to  GAPDH  expression and compared to the expression levels of hBM-MSCs. Data are expressed as mean ± SD. n = 4. ( F ) Microscopy measurement of the interaction process between allogeneic peripheral blood mononuclear cells (PBMCs) isolated from two different volunteers without or with mesenchymal stromal/stem cells (MSCs). Scale bar, 100 μm. ( G ) Comparison of immune cell proliferation during MLR assay. Autologous, allogenic, and allogenic reactions after treatment with hBM-MSCs, hUC-MSCs or three hLD-SCs, respectively, were compared. The proliferation index was calculated by dividing absorbance of 96 h co-cultured cells at a wavelength of 450 nm to that of cells before treatment.  P -values
    Figure Legend Snippet: Comparison of the characteristics between human liver-derived stem cells (hLD-SCs) and mesenchymal stromal/stem cells (MSCs) from other origins. ( A ) Microscopy imaging of human umbilical cord matrix derived stem cell (hUC-MSCs), human bone marrow stem cell (hBM-MSCs), and human liver-derived stem cells (hLD-SCs) isolated from three different individuals. Scale bar, 200 μm. ( B ) Flow cytometry measurement of hUC-MSCs, hBM-MSCs, and three hLD-SCs. CD34 was labeled as a hematopoietic stem cell marker. CD90 and CD105 were labeled as mesenchymal stem cell markers. ( C – E ) Quantitative real-time PCR (qRT-PCR) analysis of ( C ) pluripotency markers ( OCT4 , NANOG , and SOX2 ), ( D ) pro-inflammatory cytokines ( TNFα , IL1β, and IL6 ), and ( E ) anti-inflammatory cytokines ( TGFβ1 and IL10 ). Gene expression levels were normalized to GAPDH expression and compared to the expression levels of hBM-MSCs. Data are expressed as mean ± SD. n = 4. ( F ) Microscopy measurement of the interaction process between allogeneic peripheral blood mononuclear cells (PBMCs) isolated from two different volunteers without or with mesenchymal stromal/stem cells (MSCs). Scale bar, 100 μm. ( G ) Comparison of immune cell proliferation during MLR assay. Autologous, allogenic, and allogenic reactions after treatment with hBM-MSCs, hUC-MSCs or three hLD-SCs, respectively, were compared. The proliferation index was calculated by dividing absorbance of 96 h co-cultured cells at a wavelength of 450 nm to that of cells before treatment. P -values

    Techniques Used: Derivative Assay, Microscopy, Imaging, Isolation, Flow Cytometry, Labeling, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Mlr Assay, Cell Culture

    22) Product Images from "Morphology of a fibrin nanocoating influences dermal fibroblast behavior"

    Article Title: Morphology of a fibrin nanocoating influences dermal fibroblast behavior

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S162644

    Relative expression of collagen I ( A ) and Fn ( B ) measured by real-time polymerase chain reaction in human dermal fibroblasts on days 4 and 7 after cell seeding on polylactic acid membranes with a fibrin structure. The membrane with fibrin covering individual fibers (F1), with fibrin covering individual fibers and forming a mesh on the membrane surface (F2), and Fn adsorbed on fibrin (+Fn). A noncoated membrane (pristine) was used as a control sample. ACTB was used as a reference gene. The arithmetic mean ± SD was calculated for each experimental group and time interval from 10 measurements made in five independent experiments. Statistical significance ( p ≤0.05) evaluated using analysis of variance with Tukey’s method is displayed above each experimental group, indicated by the abbreviation of the group or by P representing pristine. Abbreviation:  Fn, fibronectin.
    Figure Legend Snippet: Relative expression of collagen I ( A ) and Fn ( B ) measured by real-time polymerase chain reaction in human dermal fibroblasts on days 4 and 7 after cell seeding on polylactic acid membranes with a fibrin structure. The membrane with fibrin covering individual fibers (F1), with fibrin covering individual fibers and forming a mesh on the membrane surface (F2), and Fn adsorbed on fibrin (+Fn). A noncoated membrane (pristine) was used as a control sample. ACTB was used as a reference gene. The arithmetic mean ± SD was calculated for each experimental group and time interval from 10 measurements made in five independent experiments. Statistical significance ( p ≤0.05) evaluated using analysis of variance with Tukey’s method is displayed above each experimental group, indicated by the abbreviation of the group or by P representing pristine. Abbreviation: Fn, fibronectin.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    23) Product Images from "Changes of DNA Damage Effect of T-2 or Deoxynivalenol Toxins during Three Weeks Exposure in Common Carp (Cyprinus carpio L.) Revealed by LORD-Q PCR"

    Article Title: Changes of DNA Damage Effect of T-2 or Deoxynivalenol Toxins during Three Weeks Exposure in Common Carp (Cyprinus carpio L.) Revealed by LORD-Q PCR

    Journal: Toxins

    doi: 10.3390/toxins13080576

    Specificity of the short ( a ) and long ( b ) fragments by agarose gel electrophoresis. Agarose gel (1.5%) electrophoresis of real-time PCR products with ethidium bromide staining; template DNS is 10 ng of template. ( a ) aAmplified products of the short fragment, the expected product size was 150 bp. Loading well 1–2 from T-2, loading well 3–4 from DON treated group (week 3). Loading well 5 is the negative control sample, loading well 6 is the size marker (GeneRuler TM  100 bp DNA ladder). ( b ) Amplified products of the long fragment, the expected product size was 3400 bp. Loading well 1 is the size marker (GeneRuler  TM  100 bp DNA ladder). Loading well 2–3 is from T-2, loading well 4–5 is from the DON treated group. Loading well 6 is the negative control sample.
    Figure Legend Snippet: Specificity of the short ( a ) and long ( b ) fragments by agarose gel electrophoresis. Agarose gel (1.5%) electrophoresis of real-time PCR products with ethidium bromide staining; template DNS is 10 ng of template. ( a ) aAmplified products of the short fragment, the expected product size was 150 bp. Loading well 1–2 from T-2, loading well 3–4 from DON treated group (week 3). Loading well 5 is the negative control sample, loading well 6 is the size marker (GeneRuler TM 100 bp DNA ladder). ( b ) Amplified products of the long fragment, the expected product size was 3400 bp. Loading well 1 is the size marker (GeneRuler TM 100 bp DNA ladder). Loading well 2–3 is from T-2, loading well 4–5 is from the DON treated group. Loading well 6 is the negative control sample.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Real-time Polymerase Chain Reaction, Staining, Negative Control, Marker, Amplification

    24) Product Images from "Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis"

    Article Title: Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics14010036

    Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
    Figure Legend Snippet: Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Techniques Used: Real-time Polymerase Chain Reaction

    Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
    Figure Legend Snippet: Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Techniques Used: Real-time Polymerase Chain Reaction

    25) Product Images from "Antimicrobial Effect of Isotretinoin Therapy on Periodontal Pathogens: A Case-Control Study"

    Article Title: Antimicrobial Effect of Isotretinoin Therapy on Periodontal Pathogens: A Case-Control Study

    Journal: Antibiotics

    doi: 10.3390/antibiotics10111286

    Bacterial detection frequencies of the four periodontal pathogens among the six study groups determined by real-time PCR. HC, healthy periodontium; HINN, subjects with healthy periodontium receiving isotretinoin (INN); GC, control subjects with gingivitis; GINN, generalized plaque-induced gingivitis; PC, control subjects with generalized periodontitis stage I; PINN, subjects with generalized periodontitis stage I receiving INN; *, statistically significant.
    Figure Legend Snippet: Bacterial detection frequencies of the four periodontal pathogens among the six study groups determined by real-time PCR. HC, healthy periodontium; HINN, subjects with healthy periodontium receiving isotretinoin (INN); GC, control subjects with gingivitis; GINN, generalized plaque-induced gingivitis; PC, control subjects with generalized periodontitis stage I; PINN, subjects with generalized periodontitis stage I receiving INN; *, statistically significant.

    Techniques Used: Real-time Polymerase Chain Reaction

    26) Product Images from "Sphingosine 1-Phosphate Mediates Adiponectin Receptor Signaling Essential For Lipid Homeostasis and Embryogenesis"

    Article Title: Sphingosine 1-Phosphate Mediates Adiponectin Receptor Signaling Essential For Lipid Homeostasis and Embryogenesis

    Journal: bioRxiv

    doi: 10.1101/2021.08.12.456080

    Role of SCD in maintenance of membrane homeostasis in human cells and in C. elegans . (A) Relative expression of fat-7 in N2, paqr-2 , sphk1 and nhr-49 mutant worms measured by qPCR. (B-C) PA 16:0) and OA (18:1) abundance (mol%) and in the PE of C. elegans grown on PA 2 mM plates. Related lipidomics are shown in Table S7. (D-E) Representative images C. elegans expressing GFP under the control of the fat-7 promoter ( fat-7p ::GFP) and the quantification of the fluorescence. N2 and paqr-2 mutant worms were grown on control plates and plates supplemented with S1P 25 µM for 72 h. (F-G) PA (16:0) and OA (18:1) abundance (mol%) and in the PC of HEK293 cells treated with vehicle and PA 200 µM ± S1P 1 µM. Related lipidomics are shown in Fig.S7G and in Table S6. (H) Average GP index from several images of NT and SCD siRNA HEK293 cells treated with vehicle and PA 200 µM ± S1P 10 µM and ± OA 200 µM. Data are represented as mean ± SEM. *p
    Figure Legend Snippet: Role of SCD in maintenance of membrane homeostasis in human cells and in C. elegans . (A) Relative expression of fat-7 in N2, paqr-2 , sphk1 and nhr-49 mutant worms measured by qPCR. (B-C) PA 16:0) and OA (18:1) abundance (mol%) and in the PE of C. elegans grown on PA 2 mM plates. Related lipidomics are shown in Table S7. (D-E) Representative images C. elegans expressing GFP under the control of the fat-7 promoter ( fat-7p ::GFP) and the quantification of the fluorescence. N2 and paqr-2 mutant worms were grown on control plates and plates supplemented with S1P 25 µM for 72 h. (F-G) PA (16:0) and OA (18:1) abundance (mol%) and in the PC of HEK293 cells treated with vehicle and PA 200 µM ± S1P 1 µM. Related lipidomics are shown in Fig.S7G and in Table S6. (H) Average GP index from several images of NT and SCD siRNA HEK293 cells treated with vehicle and PA 200 µM ± S1P 10 µM and ± OA 200 µM. Data are represented as mean ± SEM. *p

    Techniques Used: Expressing, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence

    S1P Signaling Via S1PR3 and SREBP1 Maintains Membrane Homeostasis in Human/Mouse Cells and in C. elegans . (A) Relative expression of S1PRs in WT MEFs measured by qPCR. (B-C) Pseudocolor images and average GP index from several images of WT MEFs treated with vehicle, PA 400 µM, PA 200 µM ± JTE-013 5 µM (S1PR2 antagonist), ± TY52156 5 µM (S1PR3 antagonist). (D) Average GP index from several images of NT, AdipoR2, S1PR2, S1PR3 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM. (E-F) Quantification of C. elegans larval stages and adulthood in N2, paqr-2 and sbp-1 mutants after 72 h growing at 20°C on normal plates in E. Tail tip morphology of 1-day adult worms grown on plates with PA-loaded OP50 bacteria in F (n=50 worms/genotype). Examples of the different tail morphologies are shown in Fig. 2T . (G) Average GP index from several images of NT, AdipoR2 and SREBF1 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM and ± OA 200 µM. (H) Western-Blot and quantification of NT and SREBF1 siRNA HEK293 cells treated with vehicle and PA 400 µM ± S1P 1 µM. The red arrow points to the precursor SREBP1 band. Note the presence of an unspecific band just below SREBP1 band. Data are represented as mean ± SD in A and ± SEM in C-D and G-H. *p
    Figure Legend Snippet: S1P Signaling Via S1PR3 and SREBP1 Maintains Membrane Homeostasis in Human/Mouse Cells and in C. elegans . (A) Relative expression of S1PRs in WT MEFs measured by qPCR. (B-C) Pseudocolor images and average GP index from several images of WT MEFs treated with vehicle, PA 400 µM, PA 200 µM ± JTE-013 5 µM (S1PR2 antagonist), ± TY52156 5 µM (S1PR3 antagonist). (D) Average GP index from several images of NT, AdipoR2, S1PR2, S1PR3 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM. (E-F) Quantification of C. elegans larval stages and adulthood in N2, paqr-2 and sbp-1 mutants after 72 h growing at 20°C on normal plates in E. Tail tip morphology of 1-day adult worms grown on plates with PA-loaded OP50 bacteria in F (n=50 worms/genotype). Examples of the different tail morphologies are shown in Fig. 2T . (G) Average GP index from several images of NT, AdipoR2 and SREBF1 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM and ± OA 200 µM. (H) Western-Blot and quantification of NT and SREBF1 siRNA HEK293 cells treated with vehicle and PA 400 µM ± S1P 1 µM. The red arrow points to the precursor SREBP1 band. Note the presence of an unspecific band just below SREBP1 band. Data are represented as mean ± SD in A and ± SEM in C-D and G-H. *p

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

    27) Product Images from "Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice"

    Article Title: Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-38609-4

    Expression of Atp2a1 (SERCA1) and Atp2a2 (SERCA2a) mRNA in EDL and soleus muscles from 4- and 12-week-old mdx and C57 control mice. Total RNA was extracted from muscles for qPCR analysis of the expression of Atp2a1 (SERCA1) and Atp2a2 (SERCA2a). Soleus ( A – D ) and EDL ( E – H ) muscle Atp2a1 (SERCA1 - A , B , E , F ) and Atp2a2 (SERCA2a - C , D , G , H ) mRNA expression from 4- and 12-week-old mdx (grey bars) and C57 control mice (white bars). N = 3–5 qPCR. Values expressed as mean ± SD. *p
    Figure Legend Snippet: Expression of Atp2a1 (SERCA1) and Atp2a2 (SERCA2a) mRNA in EDL and soleus muscles from 4- and 12-week-old mdx and C57 control mice. Total RNA was extracted from muscles for qPCR analysis of the expression of Atp2a1 (SERCA1) and Atp2a2 (SERCA2a). Soleus ( A – D ) and EDL ( E – H ) muscle Atp2a1 (SERCA1 - A , B , E , F ) and Atp2a2 (SERCA2a - C , D , G , H ) mRNA expression from 4- and 12-week-old mdx (grey bars) and C57 control mice (white bars). N = 3–5 qPCR. Values expressed as mean ± SD. *p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    28) Product Images from "Evolutionarily conserved long-chain Acyl-CoA synthetases regulate membrane composition and fluidity"

    Article Title: Evolutionarily conserved long-chain Acyl-CoA synthetases regulate membrane composition and fluidity

    Journal: eLife

    doi: 10.7554/eLife.47733

    Effect of ACSL1 silencing on UPR activation, viability, and expression of other ACSLs. ( A ) Relative expression levels measured using qPCR of several UPR markers in HEK293 cells challenged with 400 μM palmitate (PA) and treated with either non-target (NT) or ACSL1 siRNA. ( B ) Viability measured using the Trypan blue staining method of HEK293 cells in basal media or challenged with 400 μM PA and treated with either NT or ACSL1 siRNA. ( C ) Relative expression levels measured using qPCR of several ACSLs in HEK293 cells challenged with 400 μM PA and treated with either NT or ACSL1 siRNA. ( D–E ) Relative expression levels of ACSLs measured using qPCR in HEK293 cells cultivated in basal media and treated with either NT ( D ) or ACSL1 siRNA ( E ) against the indicated genes. ( F ) Change in ACSL1 expression levels in HEK293 cells cultivates din basal media and treated with siRNA against the indicated targets. Statistically significant differences of interest are indicated, where *: p
    Figure Legend Snippet: Effect of ACSL1 silencing on UPR activation, viability, and expression of other ACSLs. ( A ) Relative expression levels measured using qPCR of several UPR markers in HEK293 cells challenged with 400 μM palmitate (PA) and treated with either non-target (NT) or ACSL1 siRNA. ( B ) Viability measured using the Trypan blue staining method of HEK293 cells in basal media or challenged with 400 μM PA and treated with either NT or ACSL1 siRNA. ( C ) Relative expression levels measured using qPCR of several ACSLs in HEK293 cells challenged with 400 μM PA and treated with either NT or ACSL1 siRNA. ( D–E ) Relative expression levels of ACSLs measured using qPCR in HEK293 cells cultivated in basal media and treated with either NT ( D ) or ACSL1 siRNA ( E ) against the indicated genes. ( F ) Change in ACSL1 expression levels in HEK293 cells cultivates din basal media and treated with siRNA against the indicated targets. Statistically significant differences of interest are indicated, where *: p

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Staining

    ACS-13 is enriched on mitochondria and important for their morphology. ( A ) Confocal image of L4 staged N2 worms carrying the Pacs-13::ACS-13 isoform a::GFP translational reporter (green) and SP12 ER marker (red). Note that ACS- 13 does not colocalizes with the ER marker. R is the Pearson correlation coefficient between the two fluorophores. ( B ) Western blot detection of the GFP proteins in the indicated strains and fractions (cytosol, microsome and mitochondria); α-tubulin was used as a cytosol marker. 8 μg of protein was loaded in each sample. Note that in Pacs-13::ACS-13 isoform a::GFP worms there is enrichment of ACS-13 in the mitochondria fractionation sample showing that ACS-13 is present mostly in the mitochondria. ( C ) Quantitative PCR showing the ACS-13 RNA expression levels in N2, paqr-2 (tm3210) and mdt15(et14) worms. ( D–E ) Confocal image and quantification of one-day old adult N2 control and acs-13(et54) mutant worms carrying the mitochondrial GFP reporter myo-3::GFP and cultivated on NGM plates. Note how the mitochondria in the acs-13 mutant are less regularly spaced and of more variable length and intensity than in the wild-type. The bright green circles are muscle cell nuclei. The graph shows the frequency of worms with at least once clear instance of a muscle cell with abnormal mitochondria morphology in control myo-3::GFP and in acs-13; myo-3::GFP worms. Statistically significant differences from control are indicated, where **: p
    Figure Legend Snippet: ACS-13 is enriched on mitochondria and important for their morphology. ( A ) Confocal image of L4 staged N2 worms carrying the Pacs-13::ACS-13 isoform a::GFP translational reporter (green) and SP12 ER marker (red). Note that ACS- 13 does not colocalizes with the ER marker. R is the Pearson correlation coefficient between the two fluorophores. ( B ) Western blot detection of the GFP proteins in the indicated strains and fractions (cytosol, microsome and mitochondria); α-tubulin was used as a cytosol marker. 8 μg of protein was loaded in each sample. Note that in Pacs-13::ACS-13 isoform a::GFP worms there is enrichment of ACS-13 in the mitochondria fractionation sample showing that ACS-13 is present mostly in the mitochondria. ( C ) Quantitative PCR showing the ACS-13 RNA expression levels in N2, paqr-2 (tm3210) and mdt15(et14) worms. ( D–E ) Confocal image and quantification of one-day old adult N2 control and acs-13(et54) mutant worms carrying the mitochondrial GFP reporter myo-3::GFP and cultivated on NGM plates. Note how the mitochondria in the acs-13 mutant are less regularly spaced and of more variable length and intensity than in the wild-type. The bright green circles are muscle cell nuclei. The graph shows the frequency of worms with at least once clear instance of a muscle cell with abnormal mitochondria morphology in control myo-3::GFP and in acs-13; myo-3::GFP worms. Statistically significant differences from control are indicated, where **: p

    Techniques Used: Marker, Western Blot, Fractionation, Real-time Polymerase Chain Reaction, RNA Expression, Mutagenesis

    29) Product Images from "TraN: A novel repressor of an Enterococcus conjugative type IV secretion system"

    Article Title: TraN: A novel repressor of an Enterococcus conjugative type IV secretion system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky671

    An alternative TraN binding site on pIP501 regulates a promoter upstream of the traN gene. ( A ) Differential scanning fluorimetry (DSF) assay of TraN without DNA, with random DNA and the original binding site (o BS) as well as with alternative binding sites identified on the pIP501 plasmid (m BS, BS 2, BS 3, BS 4). For ‘no DNA’ n = 9, for all others n = 10. For a detailed sequence alignment, please see Supplementary Figure S6A . ( B ) β-galactosidase assay to monitor promoter activity in E. coli . The β-galactosidase gene was set under the control of either the P tra promoter with / without the original TraN binding site (o BS) or the P traNO promoter with / without the alternative specific binding site (BS 3). TraN or the empty vector control were supplied in trans . Enzymatic activity was assessed in E. coli with a β-galactosidase assay and Miller units are given as fold of the respective empty vector control. n = 6. ( C ) Ratio of traO mRNA levels to selected tra -gene mRNA levels ( traJ, traK and traM ). Wild type (pIP501) and mutant cells (pIP501Δ traN ) were compared to assess the activity of the potential P traNO promoter and its regulation by TraN by qPCR. mRNA levels were normalized to pyrroline-5-carboxylate reductase ( proC ) as housekeeping gene and the ratio of gene expression is given. n = 3. Mean (square) and median (center line) are depicted. For box plots, whiskers show minima and maxima within 2.2 interquartile range (IQR), for dot plots standard error of mean (s.e.m.) is given. n.s. not significant, * P
    Figure Legend Snippet: An alternative TraN binding site on pIP501 regulates a promoter upstream of the traN gene. ( A ) Differential scanning fluorimetry (DSF) assay of TraN without DNA, with random DNA and the original binding site (o BS) as well as with alternative binding sites identified on the pIP501 plasmid (m BS, BS 2, BS 3, BS 4). For ‘no DNA’ n = 9, for all others n = 10. For a detailed sequence alignment, please see Supplementary Figure S6A . ( B ) β-galactosidase assay to monitor promoter activity in E. coli . The β-galactosidase gene was set under the control of either the P tra promoter with / without the original TraN binding site (o BS) or the P traNO promoter with / without the alternative specific binding site (BS 3). TraN or the empty vector control were supplied in trans . Enzymatic activity was assessed in E. coli with a β-galactosidase assay and Miller units are given as fold of the respective empty vector control. n = 6. ( C ) Ratio of traO mRNA levels to selected tra -gene mRNA levels ( traJ, traK and traM ). Wild type (pIP501) and mutant cells (pIP501Δ traN ) were compared to assess the activity of the potential P traNO promoter and its regulation by TraN by qPCR. mRNA levels were normalized to pyrroline-5-carboxylate reductase ( proC ) as housekeeping gene and the ratio of gene expression is given. n = 3. Mean (square) and median (center line) are depicted. For box plots, whiskers show minima and maxima within 2.2 interquartile range (IQR), for dot plots standard error of mean (s.e.m.) is given. n.s. not significant, * P

    Techniques Used: Binding Assay, Plasmid Preparation, Sequencing, Activity Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing

    30) Product Images from "Effective lung-targeted RNAi in mice with peptide-based delivery of nucleic acid"

    Article Title: Effective lung-targeted RNAi in mice with peptide-based delivery of nucleic acid

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56455-2

    Nucleic acid therapy with siTNF inhibits asthma. ( a ) Schema and experimental plan of OVA/ALUM-induced asthma model, treatment with the test compounds and tissue analysis time points. Asthma was induced by sensitizing the animals with foreign protein (ovalbumin) and an adjuvant (aluminum hydroxide). The treatment consisted of systemic RNAi against TNFα, accomplished via our peptide carrier vectors PF14 and NF55. Treatment efficacy was estimated from qPCR and pathological assessment of the lung tissues. ( b ) Knockdown of TNFα in the lungs of asthmatic mice, qPCR. 100% represent treatments with either naked, nonvectorized siTNF or pshTNF. Stars represent post-hoc comparisons after one-way ANOVA F(5, 24) = 3.5, p
    Figure Legend Snippet: Nucleic acid therapy with siTNF inhibits asthma. ( a ) Schema and experimental plan of OVA/ALUM-induced asthma model, treatment with the test compounds and tissue analysis time points. Asthma was induced by sensitizing the animals with foreign protein (ovalbumin) and an adjuvant (aluminum hydroxide). The treatment consisted of systemic RNAi against TNFα, accomplished via our peptide carrier vectors PF14 and NF55. Treatment efficacy was estimated from qPCR and pathological assessment of the lung tissues. ( b ) Knockdown of TNFα in the lungs of asthmatic mice, qPCR. 100% represent treatments with either naked, nonvectorized siTNF or pshTNF. Stars represent post-hoc comparisons after one-way ANOVA F(5, 24) = 3.5, p

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay

    31) Product Images from "AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin [S]"

    Article Title: AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M092494

    The AdipoRs maintain membrane fluidity and desaturase expression in primary human cells (HUVECs). A: qPCR results showing the efficiency of the knockdown in HUVECs using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. B: Pseudocolor images showing the Laurdan dye GP index at each pixel position in HUVECs challenged with 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA. Note the pronounced rigidification of the plasma membrane in the AdipoR1/2 siRNA-treated cells. C: Average GP index from several images as in panel A ( n = 10–15). D: Distribution of the GP index values in representative images for each treatment. E: qPCR results showing the expression of three desaturases in HEK293 cells following knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. Error bars show the SDs. ** P
    Figure Legend Snippet: The AdipoRs maintain membrane fluidity and desaturase expression in primary human cells (HUVECs). A: qPCR results showing the efficiency of the knockdown in HUVECs using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. B: Pseudocolor images showing the Laurdan dye GP index at each pixel position in HUVECs challenged with 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA. Note the pronounced rigidification of the plasma membrane in the AdipoR1/2 siRNA-treated cells. C: Average GP index from several images as in panel A ( n = 10–15). D: Distribution of the GP index values in representative images for each treatment. E: qPCR results showing the expression of three desaturases in HEK293 cells following knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. Error bars show the SDs. ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    FRAP analysis showing that AdipoR1 and AdipoR2 redundantly maintain membrane fluidity in HEK293 cells. A: qPCR results showing the efficiency of the knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. B–D: FRAP results in HEK293 cells challenged with 200 μM PA and treated with NT, AdipoR1, AdipoR2, or AdipoR1/2 siRNA ( n = 6–14). E: Average T half values (the time by which half of the maximum fluorescence recovery is reached) from multiple experiments as in panels B–D. F: FRAP results in HEK293 cells challenged with 50 μM PA and treated with NT, AdipoR2, or AdipoR1/2 siRNA ( n = 10–13). G: Average T half values from panel F. H, I: FRAP results in HEK293 cells challenged with 200 μM PA and treated with either vehicle (DMSO) or 5 μM EPA ( n = 10). Error bars show the SDs in histograms and SEMs in FRAP panels. * P
    Figure Legend Snippet: FRAP analysis showing that AdipoR1 and AdipoR2 redundantly maintain membrane fluidity in HEK293 cells. A: qPCR results showing the efficiency of the knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value. B–D: FRAP results in HEK293 cells challenged with 200 μM PA and treated with NT, AdipoR1, AdipoR2, or AdipoR1/2 siRNA ( n = 6–14). E: Average T half values (the time by which half of the maximum fluorescence recovery is reached) from multiple experiments as in panels B–D. F: FRAP results in HEK293 cells challenged with 50 μM PA and treated with NT, AdipoR2, or AdipoR1/2 siRNA ( n = 10–13). G: Average T half values from panel F. H, I: FRAP results in HEK293 cells challenged with 200 μM PA and treated with either vehicle (DMSO) or 5 μM EPA ( n = 10). Error bars show the SDs in histograms and SEMs in FRAP panels. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Fluorescence

    The AdipoRs are required to maintain MUFA levels in PCs, sustain desaturase gene expression, and prevent lipotoxicity by PA. A, B: SFA and MUFA abundance (mol.%) in the PCs of HEK293 cells cultivated in the presence of either vehicle (DMSO) or 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA ( n = 3). C: qPCR results showing the expression of three desaturases in HEK293 cells following knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value . D: Percentage of dead HEK293 cells (trypan blue-positive) following cultivation in the presence of either vehicle (DMSO) or 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA. Error bars show the SDs. * P
    Figure Legend Snippet: The AdipoRs are required to maintain MUFA levels in PCs, sustain desaturase gene expression, and prevent lipotoxicity by PA. A, B: SFA and MUFA abundance (mol.%) in the PCs of HEK293 cells cultivated in the presence of either vehicle (DMSO) or 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA ( n = 3). C: qPCR results showing the expression of three desaturases in HEK293 cells following knockdown using NT, AdipoR1, and/or AdipoR2 siRNA. The expression levels are normalized to the NT value . D: Percentage of dead HEK293 cells (trypan blue-positive) following cultivation in the presence of either vehicle (DMSO) or 200 μM PA and treated with NT, AdipoR1, and/or AdipoR2 siRNA. Error bars show the SDs. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    32) Product Images from "Changes of DNA Damage Effect of T-2 or Deoxynivalenol Toxins during Three Weeks Exposure in Common Carp (Cyprinus carpio L.) Revealed by LORD-Q PCR"

    Article Title: Changes of DNA Damage Effect of T-2 or Deoxynivalenol Toxins during Three Weeks Exposure in Common Carp (Cyprinus carpio L.) Revealed by LORD-Q PCR

    Journal: Toxins

    doi: 10.3390/toxins13080576

    Specificity of the short ( a ) and long ( b ) fragments by agarose gel electrophoresis. Agarose gel (1.5%) electrophoresis of real-time PCR products with ethidium bromide staining; template DNS is 10 ng of template. ( a ) aAmplified products of the short fragment, the expected product size was 150 bp. Loading well 1–2 from T-2, loading well 3–4 from DON treated group (week 3). Loading well 5 is the negative control sample, loading well 6 is the size marker (GeneRuler TM  100 bp DNA ladder). ( b ) Amplified products of the long fragment, the expected product size was 3400 bp. Loading well 1 is the size marker (GeneRuler  TM  100 bp DNA ladder). Loading well 2–3 is from T-2, loading well 4–5 is from the DON treated group. Loading well 6 is the negative control sample.
    Figure Legend Snippet: Specificity of the short ( a ) and long ( b ) fragments by agarose gel electrophoresis. Agarose gel (1.5%) electrophoresis of real-time PCR products with ethidium bromide staining; template DNS is 10 ng of template. ( a ) aAmplified products of the short fragment, the expected product size was 150 bp. Loading well 1–2 from T-2, loading well 3–4 from DON treated group (week 3). Loading well 5 is the negative control sample, loading well 6 is the size marker (GeneRuler TM 100 bp DNA ladder). ( b ) Amplified products of the long fragment, the expected product size was 3400 bp. Loading well 1 is the size marker (GeneRuler TM 100 bp DNA ladder). Loading well 2–3 is from T-2, loading well 4–5 is from the DON treated group. Loading well 6 is the negative control sample.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Real-time Polymerase Chain Reaction, Staining, Negative Control, Marker, Amplification

    33) Product Images from "Mechanisms of Energy Metabolism in Skeletal Muscle Mitochondria Following Radiation Exposure"

    Article Title: Mechanisms of Energy Metabolism in Skeletal Muscle Mitochondria Following Radiation Exposure

    Journal: Cells

    doi: 10.3390/cells8090950

    Increased mtDNA content and expression of mitochondrial biogenesis-related genes in response to radiation exposure: ( A , B ) mtDNA content in C2C12 myotubes of control and 6-Gy IR-treated mice after 24 h, analyzed by assessing the relative levels of ND2 and gDNA by conventional PCR (A) and qPCR (B). ( C ) Representative immunoblot for COXIV and α-actin in control and 6-Gy IR-treated C2C12 myotubes after 24 h. ( D ) COXIV content quantified using image J software. ( E ) Fixed C2C12 myotubes were stained with an antibody against COXIV (green): PI (red) was used to stain the nuclei. Scale bar: 10 μm. ( F ) COXIV (green) content was quantified using image J software and normalized to that in control C2C12 myotubes. ( G ) Mitochondria content in control and 6-Gy IR-treated C2C12 myotubes after 24 h, quantified using the mitochondrial marker Mito Tracker Green. ( H ) Conventional PCR analysis of ACC-1, ACC-2, Glut-1, Glut-4, PGC-1, CPT-1, and UCP-2 mRNA in control and 6-Gy IR-treated C2C12 myotubes at the indicated times. Relative expression values were quantified using image J software and normalized to those in control C2C12 myotubes. Values are expressed as means ± SD ( n = 3; *** P
    Figure Legend Snippet: Increased mtDNA content and expression of mitochondrial biogenesis-related genes in response to radiation exposure: ( A , B ) mtDNA content in C2C12 myotubes of control and 6-Gy IR-treated mice after 24 h, analyzed by assessing the relative levels of ND2 and gDNA by conventional PCR (A) and qPCR (B). ( C ) Representative immunoblot for COXIV and α-actin in control and 6-Gy IR-treated C2C12 myotubes after 24 h. ( D ) COXIV content quantified using image J software. ( E ) Fixed C2C12 myotubes were stained with an antibody against COXIV (green): PI (red) was used to stain the nuclei. Scale bar: 10 μm. ( F ) COXIV (green) content was quantified using image J software and normalized to that in control C2C12 myotubes. ( G ) Mitochondria content in control and 6-Gy IR-treated C2C12 myotubes after 24 h, quantified using the mitochondrial marker Mito Tracker Green. ( H ) Conventional PCR analysis of ACC-1, ACC-2, Glut-1, Glut-4, PGC-1, CPT-1, and UCP-2 mRNA in control and 6-Gy IR-treated C2C12 myotubes at the indicated times. Relative expression values were quantified using image J software and normalized to those in control C2C12 myotubes. Values are expressed as means ± SD ( n = 3; *** P

    Techniques Used: Expressing, Mouse Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Software, Staining, Marker, Pyrolysis Gas Chromatography

    Radiation exposure altered the expression of mitochondrial biogenesis-related genes and mitochondrial mass in skeletal muscle: ( A ) qPCR analysis of ACC1, ACC2, Glut1, Glut4, PGC-1, CPT-1, and UCP-2 mRNA in skeletal muscle of control and (24 h after) 2-Gy IR-treated mice. Relative expression values were normalized to those in control mice. Data are presented as means ± SD ( n = 4; P
    Figure Legend Snippet: Radiation exposure altered the expression of mitochondrial biogenesis-related genes and mitochondrial mass in skeletal muscle: ( A ) qPCR analysis of ACC1, ACC2, Glut1, Glut4, PGC-1, CPT-1, and UCP-2 mRNA in skeletal muscle of control and (24 h after) 2-Gy IR-treated mice. Relative expression values were normalized to those in control mice. Data are presented as means ± SD ( n = 4; P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Mouse Assay

    34) Product Images from "The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/β-catenin dependent"

    Article Title: The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/β-catenin dependent

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.188987

    Tcf7l2 (Tcf4) is required for development of Stat3-responsive cells of zebrafish larvae intestine and the Stat3 pathway is activated ectopically in intestinal adenomas of apc hu745 mutants. (A,A′) In vivo EGFP fluorescence in the intestine of 6 dpf Tg(7xStat3:EGFP) / tcf7l2 hu892 / hu892 , Tg(7xStat3:EGFP) / tcf7l2 +/ hu892 and Tg(7xStat3:EGFP) / tcf7l2 +/+ siblings (A) and measurement of integrated density (A′) ( n =16). (B) qPCR analysis of il6 , gp130 , jak2a , jak2b and stat3 mRNA expression from tcf7l2 +/+ and tcf7l2 hu892/hu892 sibling larvae. (C,C′) Effect of XAV treatment on Tg(7xStat3:EGFP) embryos from 48 to 78 hpf: measurement of the integrated density of the fluorescence (C) and measurement of the number of GFP + cells (C′). (D,E) Haematoxylin-eosin staining on paraffin embedded transversal section of zebrafish apc hu745 intestine at 12 months post fertilization, displaying both normal tissue (n) and hyperplastic adenomas (a). (D′,D″) Staining of a sequential intestinal section of D using α-EGFP (green) (D′) and α-PCNA (red) (D″) Abs. (E′,E″) Double staining using α-EGFP Ab (green) and α-PCNA Ab (red) of a sequential intestinal section of E. All statistical analyses were performed by unpaired t -test. * P
    Figure Legend Snippet: Tcf7l2 (Tcf4) is required for development of Stat3-responsive cells of zebrafish larvae intestine and the Stat3 pathway is activated ectopically in intestinal adenomas of apc hu745 mutants. (A,A′) In vivo EGFP fluorescence in the intestine of 6 dpf Tg(7xStat3:EGFP) / tcf7l2 hu892 / hu892 , Tg(7xStat3:EGFP) / tcf7l2 +/ hu892 and Tg(7xStat3:EGFP) / tcf7l2 +/+ siblings (A) and measurement of integrated density (A′) ( n =16). (B) qPCR analysis of il6 , gp130 , jak2a , jak2b and stat3 mRNA expression from tcf7l2 +/+ and tcf7l2 hu892/hu892 sibling larvae. (C,C′) Effect of XAV treatment on Tg(7xStat3:EGFP) embryos from 48 to 78 hpf: measurement of the integrated density of the fluorescence (C) and measurement of the number of GFP + cells (C′). (D,E) Haematoxylin-eosin staining on paraffin embedded transversal section of zebrafish apc hu745 intestine at 12 months post fertilization, displaying both normal tissue (n) and hyperplastic adenomas (a). (D′,D″) Staining of a sequential intestinal section of D using α-EGFP (green) (D′) and α-PCNA (red) (D″) Abs. (E′,E″) Double staining using α-EGFP Ab (green) and α-PCNA Ab (red) of a sequential intestinal section of E. All statistical analyses were performed by unpaired t -test. * P

    Techniques Used: In Vivo, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, Staining, Double Staining

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    Solis BioDyne hot firepol evagreen qpcr supermix
    Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean <t>−qPCR</t> Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.
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    Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Journal: Pharmaceutics

    Article Title: Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

    doi: 10.3390/pharmaceutics14010036

    Figure Lengend Snippet: Impact of the AZT-liposomes on the growth of C. trachomatis serovar F clinical isolate. ( A ) Direct C. trachomatis growth in the presence of free AZT and encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Article Snippet: Briefly, the composition of the reaction included 2 μL 5× HOT FIREPol® EvaGreen® qPCR Supermix, 1–1 μL forward and reverse primers (10 pmol each), 1 μL template and 5 µL MQ water.

    Techniques: Real-time Polymerase Chain Reaction

    Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Journal: Pharmaceutics

    Article Title: Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

    doi: 10.3390/pharmaceutics14010036

    Figure Lengend Snippet: Impact of different types of AZT-liposomes on the growth of C. trachomatis serovar D. ( A ) Direct C. trachomatis growth in the presence of free AZT and liposomally-encapsulated AZT. ( B ) Recoverable C. trachomatis growth. Data are mean −qPCR Ct levels ± S.D. ( n = 3). EL-AZT, neutral liposomes with azithromycin; +EL-AZT, cationic liposomes with azithromycin; −EL-AZT, anionic liposomes with azithromycin.

    Article Snippet: Briefly, the composition of the reaction included 2 μL 5× HOT FIREPol® EvaGreen® qPCR Supermix, 1–1 μL forward and reverse primers (10 pmol each), 1 μL template and 5 µL MQ water.

    Techniques: Real-time Polymerase Chain Reaction

    BAPTA-AM induced sensitization by sub-toxic-dose of cisplatin is associated with a decrease in mRNA expression of p21, calmodulin, S100A8, and S100A14. A. Cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, in the presence or absence of BAPTA-AM. After treatment, total RNA was extracted from cells followed by cDNA synthesis and real-time qPCR. All mRNA expression levels were normalized against GAPDH. The data show the normalized expression of (A) p21, (B) calmodulin, (C) S100A8, and (D) S100A14, relative to the expression of the control. Each value is the mean ± SEM. Experiments were conducted with triplicate samples and repeated at least twice. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test, ∗ P

    Journal: Heliyon

    Article Title: Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells

    doi: 10.1016/j.heliyon.2021.e06041

    Figure Lengend Snippet: BAPTA-AM induced sensitization by sub-toxic-dose of cisplatin is associated with a decrease in mRNA expression of p21, calmodulin, S100A8, and S100A14. A. Cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, in the presence or absence of BAPTA-AM. After treatment, total RNA was extracted from cells followed by cDNA synthesis and real-time qPCR. All mRNA expression levels were normalized against GAPDH. The data show the normalized expression of (A) p21, (B) calmodulin, (C) S100A8, and (D) S100A14, relative to the expression of the control. Each value is the mean ± SEM. Experiments were conducted with triplicate samples and repeated at least twice. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test, ∗ P

    Article Snippet: Real-time quantitative PCR was performed with 5x HOT FIREPol® EvaGreen® qPCR Supermix kit (Solis BioDyne, Tartu, Estonia) using StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Differential and dose-dependent effect of cisplatin on mRNA expression levels of p21, calmodulin, S100A8, and S100A14. After cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, mRNA expression levels were quantified by qPCR. All mRNA expression levels were normalized against GAPDH. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test. Each value is the mean ± SEM from three independent experiments, each with three replicates. ∗ P

    Journal: Heliyon

    Article Title: Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells

    doi: 10.1016/j.heliyon.2021.e06041

    Figure Lengend Snippet: Differential and dose-dependent effect of cisplatin on mRNA expression levels of p21, calmodulin, S100A8, and S100A14. After cells were exposed to sub-toxic (20 μM), 40 μM, and 100 μM of cisplatin for 18 h, mRNA expression levels were quantified by qPCR. All mRNA expression levels were normalized against GAPDH. Statistically significant differences were determined by one-way ANOVA. Statistical comparison between two groups was performed using t -test. Each value is the mean ± SEM from three independent experiments, each with three replicates. ∗ P

    Article Snippet: Real-time quantitative PCR was performed with 5x HOT FIREPol® EvaGreen® qPCR Supermix kit (Solis BioDyne, Tartu, Estonia) using StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Growth and chlorophyll fluorescence phenotypes of the lpa2 mutant compared to wild type and complemented lines. A, Alignment of LPA2 amino acid sequences from different organisms. Predicted chloroplast transit peptides are shown in grey, predicted transmembrane helices are underlined and indicated with pipes. Peptides identified by mass spectrometry are given in red letters, twin-arginines in bold letters. Ath – Arabidopsis thaliana (AT5G51545), Zma – Zea mays (NP_001145487), Psi – Picea sitchensis (ABK23742), Ppa – Physcomitrella patens (XP_024366975), Cva – Chlorella variabilis (XP_005849843), Ota – Ostreococcus tauri (XP_003084445), Cre – Chlamydomonas reinhardtii (Cre02.g105650). B, Structure of the Chlamydomonas LPA2 gene, insertion site of the CIB1 cassette in the lpa2 mutant, and construct for complementation. Protein coding regions are drawn as black boxes, untranslated regions as bars, and introns and promoter regions as thin lines. Arrows indicate transcriptional start sites. The purple box indicates a 165-bp fragment derived from the 19th intron of gene Cre13.g573450 in reverse orientation that has integrated together with the CIB1 cassette. C, qRT-PCR analysis of LPA2 transcript accumulation. Values are means from two independent biological replicates and indicate LPA2 transcript levels in the lpa2 mutant and two complemented lines expressing the LPA2 cDNA without (c10, c11) or with a 3xHA coding region (cHA) relative to the wild type. Error bars indicate SD. D, Comparison of PSII maximum quantum efficiency (Fv/Fm). Shown are averages from 3-6 independent experiments, error bars indicate SD. Significant differences were assessed via T-test, (*** p

    Journal: bioRxiv

    Article Title: Complexome profiling on the lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins

    doi: 10.1101/2021.01.04.425283

    Figure Lengend Snippet: Growth and chlorophyll fluorescence phenotypes of the lpa2 mutant compared to wild type and complemented lines. A, Alignment of LPA2 amino acid sequences from different organisms. Predicted chloroplast transit peptides are shown in grey, predicted transmembrane helices are underlined and indicated with pipes. Peptides identified by mass spectrometry are given in red letters, twin-arginines in bold letters. Ath – Arabidopsis thaliana (AT5G51545), Zma – Zea mays (NP_001145487), Psi – Picea sitchensis (ABK23742), Ppa – Physcomitrella patens (XP_024366975), Cva – Chlorella variabilis (XP_005849843), Ota – Ostreococcus tauri (XP_003084445), Cre – Chlamydomonas reinhardtii (Cre02.g105650). B, Structure of the Chlamydomonas LPA2 gene, insertion site of the CIB1 cassette in the lpa2 mutant, and construct for complementation. Protein coding regions are drawn as black boxes, untranslated regions as bars, and introns and promoter regions as thin lines. Arrows indicate transcriptional start sites. The purple box indicates a 165-bp fragment derived from the 19th intron of gene Cre13.g573450 in reverse orientation that has integrated together with the CIB1 cassette. C, qRT-PCR analysis of LPA2 transcript accumulation. Values are means from two independent biological replicates and indicate LPA2 transcript levels in the lpa2 mutant and two complemented lines expressing the LPA2 cDNA without (c10, c11) or with a 3xHA coding region (cHA) relative to the wild type. Error bars indicate SD. D, Comparison of PSII maximum quantum efficiency (Fv/Fm). Shown are averages from 3-6 independent experiments, error bars indicate SD. Significant differences were assessed via T-test, (*** p

    Article Snippet: Quantitative reverse transcription-PCR (qRT-PCR) was performed using the StepOnePlus RT-PCR system (Applied Biosystems) and the 5x HOT FIREPol® EvaGreen® qPCR Supermix kit from Solis BioDyne.

    Techniques: Fluorescence, Mutagenesis, Mass Spectrometry, Construct, Derivative Assay, Quantitative RT-PCR, Expressing

    Importance of AdipoR2 to regulate membrane homeostasis. A , qPCR results showing the efficiency of different knockdowns. B , Average T half values from FRAP experiments comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 0 h PA (NT siRNA, n=7; AdipoR2 siRNA, n=6; SCD siRNA, n=5; FADS2 siRNA, n=6; ACSL4 siRNA, n=6; PEMT siRNA, n=6). C , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, siRNA after 3 h 200 μM PA (NT siRNA, n=6; AdipoR2 siRNA, n=7; SCD siRNA, n=5; FADS2 siRNA, n=5). D , E , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 6 h PA (NT siRNA, n=8; AdipoR2 siRNA, n=15; SCD siRNA, n=9; FADS2 siRNA, n=10; ACSL4 siRNA, n=8; PEMT siRNA, n=8). F , G , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 15 h PA (NT siRNA, n=5; AdipoR2 siRNA, n=13; SCD siRNA, n=11; FADS2 siRNA, n=10; ACSL4 siRNA, n=9; PEMT siRNA, n=5). H , I , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 24 h 200 μM PA c (NT siRNA, n=10; AdipoR2 siRNA, n=15; SCD siRNA, n=15; FADS2 siRNA, n=15; ACSL4 siRNA, n=15; PEMT siRNA, n=10). J , Distribution of GP index values for cells treated with NT, AdipoR2, SCD, FADS2 siRNA and challenged with PA. K-N , MUFAs and PUFAs abundance (mol%) in PCs and PEs in cells treated with different siRNAs and PA (n=4 for all treatments). O-R , PC/PE ratio, relative lysophosphatidylcholine (LPC), free cholesterol (FC) and total TAGs in cells treated with different siRNAs and PA (n=4 for all treatments). S-U , Relative gene expression for ATF4, DDiT3 and HSPA5 in cells treated different siRNAs and PA (n=4 for all treatments).

    Journal: bioRxiv

    Article Title: AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

    doi: 10.1101/2020.06.11.144329

    Figure Lengend Snippet: Importance of AdipoR2 to regulate membrane homeostasis. A , qPCR results showing the efficiency of different knockdowns. B , Average T half values from FRAP experiments comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 0 h PA (NT siRNA, n=7; AdipoR2 siRNA, n=6; SCD siRNA, n=5; FADS2 siRNA, n=6; ACSL4 siRNA, n=6; PEMT siRNA, n=6). C , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, siRNA after 3 h 200 μM PA (NT siRNA, n=6; AdipoR2 siRNA, n=7; SCD siRNA, n=5; FADS2 siRNA, n=5). D , E , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 6 h PA (NT siRNA, n=8; AdipoR2 siRNA, n=15; SCD siRNA, n=9; FADS2 siRNA, n=10; ACSL4 siRNA, n=8; PEMT siRNA, n=8). F , G , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 15 h PA (NT siRNA, n=5; AdipoR2 siRNA, n=13; SCD siRNA, n=11; FADS2 siRNA, n=10; ACSL4 siRNA, n=9; PEMT siRNA, n=5). H , I , Average T half values comparing cells treated with NT, AdipoR2, SCD, FADS2, ACSL4 and PEMT siRNA after 24 h 200 μM PA c (NT siRNA, n=10; AdipoR2 siRNA, n=15; SCD siRNA, n=15; FADS2 siRNA, n=15; ACSL4 siRNA, n=15; PEMT siRNA, n=10). J , Distribution of GP index values for cells treated with NT, AdipoR2, SCD, FADS2 siRNA and challenged with PA. K-N , MUFAs and PUFAs abundance (mol%) in PCs and PEs in cells treated with different siRNAs and PA (n=4 for all treatments). O-R , PC/PE ratio, relative lysophosphatidylcholine (LPC), free cholesterol (FC) and total TAGs in cells treated with different siRNAs and PA (n=4 for all treatments). S-U , Relative gene expression for ATF4, DDiT3 and HSPA5 in cells treated different siRNAs and PA (n=4 for all treatments).

    Article Snippet: Quantitative PCR (qPCR) Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND-1000; ThermoFisher). cDNA was obtained using a RevertAid H Minus First Strand cDNA Synthesis Kit with random hexamers (ThermoFisher). qPCR experiments were performed with a CFX Connect thermal cycler (Bio Rad) using Hot FIREpol EvaGreen qPCR SuperMix (Solis Biodyne) and standard primers.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    AdipoR2 regulates membrane homeostasis to counter PA-induced lipotoxicity. A , Differential T half values comparing AdipoR2, SCD, FADS2, ACSL4 and PEMT knockdown (KD) in HEK293 cells challenged with 200 μM PA in a time-course FRAP experiment. B , Representative pseudocolor images and C , average laurdan dye GP index in HEK293 cells treated with AdipoR2, ACSL4 or FADS2 siRNA and challenged with 200 μM PA (n=15 for all treatments). D , E and F, SFAs abundance (mol%) in PCs and PEs and relative levels of TAG 48:0 in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). G , Spliced XBP1 mRNA expression in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). H , Efficiency of AdipoR2 silencing in HUVECs measured using qPCR. I , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT (nontarget) and AdipoR2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; AdipoR2 siRNA, n=3). J , AdipoR2-dependent membrane regulation to counter SFA-induced lipotoxicity: Dietary SFAs are taken up by the cells and become incorporated into membrane phospholipids (1), leading to plasma membrane rigidification (2), ER stress (3), and impairment of mitochondria function (4). In healthy cells, AdipoR2 responds to increased membrane rigidification by signalling to promote the expression of desaturases and other lipid metabolism genes (5), leading to increased levels of UFAs available for incorporation into phospholipids (6) and normalization of membrane fluidity (7). Some cells can also sequester SFAs into lipid droplets in the form of TAGs (8). A similar process occurs in C. elegans , where rigidification activates the AdipoR homolog PAQR-2 by promoting its interaction with IGLR-2.

    Journal: bioRxiv

    Article Title: AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

    doi: 10.1101/2020.06.11.144329

    Figure Lengend Snippet: AdipoR2 regulates membrane homeostasis to counter PA-induced lipotoxicity. A , Differential T half values comparing AdipoR2, SCD, FADS2, ACSL4 and PEMT knockdown (KD) in HEK293 cells challenged with 200 μM PA in a time-course FRAP experiment. B , Representative pseudocolor images and C , average laurdan dye GP index in HEK293 cells treated with AdipoR2, ACSL4 or FADS2 siRNA and challenged with 200 μM PA (n=15 for all treatments). D , E and F, SFAs abundance (mol%) in PCs and PEs and relative levels of TAG 48:0 in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). G , Spliced XBP1 mRNA expression in HEK293 cells with the indicated genes knocked down and challenged with 200 μM PA (n=4 for all treatments). H , Efficiency of AdipoR2 silencing in HUVECs measured using qPCR. I , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT (nontarget) and AdipoR2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; AdipoR2 siRNA, n=3). J , AdipoR2-dependent membrane regulation to counter SFA-induced lipotoxicity: Dietary SFAs are taken up by the cells and become incorporated into membrane phospholipids (1), leading to plasma membrane rigidification (2), ER stress (3), and impairment of mitochondria function (4). In healthy cells, AdipoR2 responds to increased membrane rigidification by signalling to promote the expression of desaturases and other lipid metabolism genes (5), leading to increased levels of UFAs available for incorporation into phospholipids (6) and normalization of membrane fluidity (7). Some cells can also sequester SFAs into lipid droplets in the form of TAGs (8). A similar process occurs in C. elegans , where rigidification activates the AdipoR homolog PAQR-2 by promoting its interaction with IGLR-2.

    Article Snippet: Quantitative PCR (qPCR) Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND-1000; ThermoFisher). cDNA was obtained using a RevertAid H Minus First Strand cDNA Synthesis Kit with random hexamers (ThermoFisher). qPCR experiments were performed with a CFX Connect thermal cycler (Bio Rad) using Hot FIREpol EvaGreen qPCR SuperMix (Solis Biodyne) and standard primers.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    AdipoR2 silencing causes rigidification measured by atomic force microscopy and also alters response to different SFAs. A , qPCR results showing the efficiency of AdipoR2 knockdown in HEK293 cells; NT stands for nontarget siRNA. B , Membrane deformation measured by atomic force microscopy in HEK293 cells treated with NT and AdipoR2 siRNA under basal conditions or treated with 200 μM PA (NT siRNA and AdipoR2 siRNA Basal, n=66; NT siRNA PA, n=66; AdipoR2 siRNA PA, n=67). C , Representative deformation curve for NT and AdipoR2 siRNA-treated cells in the presence of PA D , The SFAs myristic acid (MA), PA and stearic acid (SA) all cause membrane rigidification in AdipoR2-knocked down (KD) HEK293 cells as measured using FRAP; the average T half is shown for each condition (n≥7).

    Journal: bioRxiv

    Article Title: AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

    doi: 10.1101/2020.06.11.144329

    Figure Lengend Snippet: AdipoR2 silencing causes rigidification measured by atomic force microscopy and also alters response to different SFAs. A , qPCR results showing the efficiency of AdipoR2 knockdown in HEK293 cells; NT stands for nontarget siRNA. B , Membrane deformation measured by atomic force microscopy in HEK293 cells treated with NT and AdipoR2 siRNA under basal conditions or treated with 200 μM PA (NT siRNA and AdipoR2 siRNA Basal, n=66; NT siRNA PA, n=66; AdipoR2 siRNA PA, n=67). C , Representative deformation curve for NT and AdipoR2 siRNA-treated cells in the presence of PA D , The SFAs myristic acid (MA), PA and stearic acid (SA) all cause membrane rigidification in AdipoR2-knocked down (KD) HEK293 cells as measured using FRAP; the average T half is shown for each condition (n≥7).

    Article Snippet: Quantitative PCR (qPCR) Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND-1000; ThermoFisher). cDNA was obtained using a RevertAid H Minus First Strand cDNA Synthesis Kit with random hexamers (ThermoFisher). qPCR experiments were performed with a CFX Connect thermal cycler (Bio Rad) using Hot FIREpol EvaGreen qPCR SuperMix (Solis Biodyne) and standard primers.

    Techniques: Microscopy, Real-time Polymerase Chain Reaction

    The SREBFs regulate membrane composition and fluidity in HEK293 cells challenged with 200 μM PA. A , qPCR results showing the efficiency of different knockdowns in HEK293 cells (n=3 for all treatments). B , Relative SCD expression in cells treated with different siRNAs (n=3 for all treatments). C , D Average T half values comparing cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA after C , 0 h PA challenge (n=6 for all treatments) and D , 24 h PA challenge (NT siRNA, n=10; SREBF1 siRNA, n=11; SREBF2 siRNA, n=10; SREBF1+2 siRNA, n=11). E , Distribution of GP index values for cells treated with SREBF1, SREBF2, SREBF1+2 siRNA and challenged with PA. F, Representative pseudocolor images of laurdan dye GP index and G, average GP index in HEK293 cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA and challenged with 200 μM PA (n=15 for all treatments). H-M , SFA, MUFA and PUFA abundance (mol%) in PCs and PEs in cells treated with different siRNAs and 200 μM PA (n=4 for all treatments). N , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT, SREBF1 and SREBF2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; SREBF1 siRNA, n=3; SREBF1 siRNA, n=3).

    Journal: bioRxiv

    Article Title: AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

    doi: 10.1101/2020.06.11.144329

    Figure Lengend Snippet: The SREBFs regulate membrane composition and fluidity in HEK293 cells challenged with 200 μM PA. A , qPCR results showing the efficiency of different knockdowns in HEK293 cells (n=3 for all treatments). B , Relative SCD expression in cells treated with different siRNAs (n=3 for all treatments). C , D Average T half values comparing cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA after C , 0 h PA challenge (n=6 for all treatments) and D , 24 h PA challenge (NT siRNA, n=10; SREBF1 siRNA, n=11; SREBF2 siRNA, n=10; SREBF1+2 siRNA, n=11). E , Distribution of GP index values for cells treated with SREBF1, SREBF2, SREBF1+2 siRNA and challenged with PA. F, Representative pseudocolor images of laurdan dye GP index and G, average GP index in HEK293 cells treated with NT, SREBF1, SREBF2 and SREBF1+2 siRNA and challenged with 200 μM PA (n=15 for all treatments). H-M , SFA, MUFA and PUFA abundance (mol%) in PCs and PEs in cells treated with different siRNAs and 200 μM PA (n=4 for all treatments). N , Relative levels of SFAs, MUFAs and PUFAs in PCs in HUVECs treated with NT, SREBF1 and SREBF2 siRNA and challenged with 200 μM PA for 6 h (NT siRNA, n=5; SREBF1 siRNA, n=3; SREBF1 siRNA, n=3).

    Article Snippet: Quantitative PCR (qPCR) Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND-1000; ThermoFisher). cDNA was obtained using a RevertAid H Minus First Strand cDNA Synthesis Kit with random hexamers (ThermoFisher). qPCR experiments were performed with a CFX Connect thermal cycler (Bio Rad) using Hot FIREpol EvaGreen qPCR SuperMix (Solis Biodyne) and standard primers.

    Techniques: Real-time Polymerase Chain Reaction, Expressing