Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Clinicopathological and experimental data obtained from patients with NSCLC from La Paz University Hospital

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Methylation, Expressing

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Involvement of miR-7 and MAFG in patients with NSCLC. (A-B) Assessment of miR-7 methylation levels measured by qMSP and expression levels of miR-7 measured by qRT-PCR in fresh samples from a cohort of 22 patients with NSCLC; (A) Left, quantitative methylation percentage of miR-7; right, miR-7 expression levels in 44 paired samples (ATT and T); (B) Correlation between the methylation percentage of miR-7 and expression levels of miR-7. For all the analyses, data represent the percentage of methylation according to a previous report and expression levels in 2 −ΔCt ; (C) Analysis of mRNA expression of MAFG in two in silico cohorts of patients with NSCLC. Survival analysis in 984 (left panel) and 1035 (right panel) NSCLC samples from the TCGA and TCC, respectively. LogRank test was used for comparisons and P < 0.05 was considered as a significant change in OS. NSCLC, non-small cell lung cancer; ATT, adjacent tumor tissue; T, tumor; CPM, counts per million; TCGA, The Cancer Genome Atlas; TCC, Total Cancer Care. *** P < 0.001

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Methylation, Expressing, Quantitative RT-PCR, In Silico

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Viability assays and reactive oxygen species detection after CDDP treatment in H23 cell lines. (A) Viability to CDDP in H23S (left) and H23R (right) at 24, 48, and 72 hours and 6 concentrations of CDDP measured by MTT assay; (B) Increment of ROS production in H23S (left) and H23R (right) after 24, 48, and 72 hours of CDDP treatment; For (A) and (B), bars represent the mean of at least two independent experiments measured by duplicate ± SD. $: P ≤ 0.05 versus basal; $$: P ≤ 0.01 versus basal; $$$: P ≤ 0.001 were considered as significant change in CDDP resistance and ROS production; (C) Semiquantitative mRNA expression analysis of downstream genes involved in ROS detoxification regulated by MAFG, which showed expression changes for H23 in the microarray data (GSE84201, ) (left panel). Representative images of MAFG and HMOX1 RT-PCR comparing sensitive and resistant subtypes. Each assay was performed at least three times to confirm the results. (Right panel) Relative intensity quantification of the amplified band for each gene measured by ImageJ Software. Bars represent the mean of the three independent experiments using the intensity of GAPDH as endogenous control and the sensitive subtype of each cell line as calibrator. (D) Immunoblots of HMOX1, NRF2 , and -actin. Images correspond to a representative experiment that was repeated four times with similar results. Scion Image program was used to quantify band intensities corresponding to immunoblot detection of protein samples. CDDP, cisplatin; MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: MTT Assay, Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Amplification, Software, Control, Western Blot

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Analysis of the affinity of the aptamers against MAFG. (A) Quantification of aptamer-MAFG complexes by qPCR comparing absence (dot pattern) or presence (filled pattern) of MAFG protein. Figure shows the differences in Threshold Cycle (Ct) between the library RND40 and the selection rounds 3 (SEL3MAFG) and 6 (SEL6MAFG). Bars represent the mean ± SEM of 2–3 experiments; (B) Specificity binding assay of aptamers to MAFG performed by ELONA. MAFG-Myc was plated at 50 ng/well in 96-well plates followed by incubation with 5' digoxigenin-labeled aptamers from RND40, SEL3MAFG, and SEL6MAFG. The figure represents the mean ± SEM of three independent experiments. $$: P < 0.01; (C) Aptacytochemistry in HEK-293T cells transiently transfected with MAFG-Myc. Cells overexpressing MAFG were incubated with 5' Alexafluor 488-conju-gated aptamers from RND40, SEL3MAFG, or SEL6MAFG (upper panel) or Myc antibody (lower panel). Fluorescence microscopy images corresponding to the staining of nuclei with Hoechst (blue), antibodies (red), and aptamers (green) are shown. Arrowhead indicates positive cells. Bar = 13 μ m; (D) Study of individual aptamer affinity performed by ELONA. Recombinant MAFG was plated at 50 ng/well in 96-well plates followed by incubation with 5' digoxigenin-labeled individual aptamers apMAGF3F, apMAFG6F, and apMAFG11F. The figure represents the mean ± SEM of two independent experiments; (E) Secondary structures of aptamers apMAGF3F, apMAFG6F, and apMAFG11F obtained by bioinformatics sequence analysis using the mFold software taking into account the lower free energy (ΔG). MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G; ELONA, enzyme-linked oligonucleotide assay.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Selection, Binding Assay, Incubation, Labeling, Transfection, Fluorescence, Microscopy, Staining, Recombinant, Sequencing, Software, Oligonucleotide Assay

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: MAFG localization pattern in H23S and H23R cells measured by aptacytochemistry. Cells were incubated using digoxigenin-labeled aptamers apMAFG3F, apMAFG6F, and apMAFG11F as primary recognition molecule and Alexafluor 488-conjugated anti-digoxigenin as secondary antibody. Confocal microscopy images corresponding to the staining of nuclei with Dapi (blue), aptamers (green) and merge are shown. Bar = 25 μ m. MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Incubation, Labeling, Confocal Microscopy, Staining

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Analysis of MAFG localization pattern in paraffin samples of patients with known levels of MAFG by aptahistochemistry. Samples were incubated with digoxigenin-labeled aptamers apMAFG3F, apMAFG6F, and apMAFG11F as primary recognition molecules, and afterward with anti-digoxigenin antibodies. Staining was performed using DAB staining solution. (A) Representative aptahistochemistry pictures showing the areas of analysis in NSCLC tumor samples; (B) T test comparison between low and high MAFG mRNA levels (2 −ΔCt ) and the % mean ratio of positive cells analyzed in the areas defined on (A). Positive cells were counted using the Cell Counter on ImageJ software. NSCLC: non-small cell lung cancer. MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Incubation, Labeling, Staining, Comparison, Software

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Effect of aptamers apMAFG3F, apMAFG6F, and apMAFG11F on cell viability, response to CDDP. (A) Viability curves combining the selected dose of aptamers (25 nM) and increasing doses of CDDP in H23S/R cells. # P < 0.05 (Student’s t test). The treatment with aptamer significantly increased the sensitivity to CDDP (* P < 0.05 and ***< 0.001); (B) Viability curves combining apMAFG6F at 25 nM with increasing doses of CDDP in H1299 lung cancer cells. (C) Viability curves of H23 cell line transfected with pCMV6 (S-Ø and R-), with the overexpression vectors (S-MAFG) and the combination of MAFG overexpressing plasmids together with apMAFG6F at 25 nM. For (A), (B), and (C), each experimental group was exposed for 72 h to 6 different CDDP concentrations after aptamer transfection; data were normalized to each untreated control, set to 100% and represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. The CDDP-RI (Resistant Index to CDDP) was calculated as “IC50 from the Resistant/ IC50 from the Sensitive" and “IC50 from the transfected with aptamers or MAFG/ IC50 from the Sensitive” ± SD. (D) Validation of MAFG overexpression and aptamer transfection in the H23S cells after 72 h of transfection. Top, Relative expression levels of MAFG measured by quantitative RT-PCR, in the cell line H23, represented in 2 −ΔΔCt scale; In each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. Bottom, quantification of apMAFG6F in the cell line H23. Quantification was performed according to the absolute quantification method by using a standard curve of known concentration of aptamers. The amount of protein per sample was used as a control. Data represent the mean of at least three independent experiments measured by triplicate ± SD. CDDP, cisplatin; RT-PCR, real-time polymerase chain reaction.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Transfection, Over Expression, Control, Concentration Assay, Biomarker Discovery, Expressing, Quantitative RT-PCR, Plasmid Preparation, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Effect of aptamer apMAFG6F on response to CDDP and ROS production. (A) Viability and ROS production assay in H23R cells after apMAFG6F transfection. Each experimental group was exposed for 72 h to 6 different CDDP concentrations, and data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least four independent experiments performed in duplicate at each drug concentration for each cell line analyzed. * P < 0.05 was considered as significant change in CDDP resistance and ROS production; (B) Expression analysis of downstream genes regulated by MAFG involved in ROS detoxification in H23R and H23R transfected with apMAFG6F. Left, representative images of MAFG , HMOX1 , and GAPDH RT-PCR. Each assay was performed at least three times to confirm the results. Right, relative intensity quantification of the amplified band for each gene measured by ImageJ Software. Bars represent the mean of three independent experiments using the intensity of GAPDH as endogenous control and the resistant subtype of each cell line as calibrator ± SD. CDDP, cisplatin; ROS, reactive oxygen species; MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Transfection, Control, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Software

Journal:

Article Title: Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

doi: 10.1128/JCM.39.7.2618-2626.2001

Figure Lengend Snippet: Specificity panel for Legionella LCR-PCR assays

Article Snippet: Control strains of Legionella table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Bacterial strain Source a Result of PCR with primer-probe set 5S rDNA mip Stenotrophomonas maltophilia CDC AB9-D19-80 − − Morganella morganii − − Bordetella bronchoseptica − − Bordetella pertussis ATCC VR1310 − − Bordetella parapertussis ATCC 27853 − − Chlamydia pneumoniae ATCC 25922 − − Pseudomonas aeruginosa − − Escherichia coli CDC B2-003-72 − − Klebsiella pneumoniae − − Streptococcus pneumoniae − − Mycoplasma pneumoniae − − Mycobacterium tuberculosis − − Mycobacterium avium complex CDC ABD-D20-82 − − Streptococcus sp. viridans group − − Listeria monocytogenes − − Staphylococcus epidermidis CDC AB4-BID-84 − − Aeromonas hydrophila − − Pseudomonas fluorescens − − Moraxella catarrhalis − − Mycoplasma pneumoniae − − Pseudomonas cepacia − − Acinetobacter sp. ATCC 25923 − − Klebsiella oxytoca − − Staphylococcus aureus CDC AB4-B08-84 − − Proteus mirabilis − − Streptococcus pyogenes ATCC 10211 − − Proteus vulgaris − − Haemophilus influenzae − − Bacteroides fragilis CDC AB2-C15-82 − − Citrobacter freundii − − Campylobacter jejuni − − Open in a separate window a If not otherwise specified, strains are Mayo Clinic isolates.

Techniques:

Journal: PLoS ONE

Article Title: Diannexin Protects against Renal Ischemia Reperfusion Injury and Targets Phosphatidylserines in Ischemic Tissue

doi: 10.1371/journal.pone.0024276

Figure Lengend Snippet: ( A ) Representative western blot of kidney homogenate from DA5 and vehicle-treated mice (ctrl), after 3 or 7 days of reperfusion, stained for KIM-1, NGAL and β-actin. Purified KIM-1 and NGAL protein were used as positive control. (B,C,D) When compared to sham operation, KIM-1 and NGAL expression, as well as KIM-1 transcription, were increased in renal tissue 3 and 8 days after IRI induction. DA5 treatment significantly reduced protein expression on day 3 (KIM-1 and NGAL) and day 8 (KIM-1), when compared to vehicle-treated mice (ctrl). Also, DA5 reduced the IRI-induced transcription of KIM-1 on day 3 and day 8. (E) NGAL mRNA expression was not significantly elevated on day 3 or day 8 post -IRI. CT-values for corresponding β-actin were 17.2±3.2 for KIM-1 and 18.5±2.9 for NGAL. Values are means ± SEM. * P< 0.05, ** P< 0.001 vs. sham group; # P< 0.05, ## P< 0.001 vs. ctrl group. n = 5 for sham operated mice and n = 10 for all other groups.

Article Snippet: SYBR Green® and Taqman® Universal PCR Master Mix and NGAL and β-actin primer probe sets (Mm01324470_a1 and 4352933E) were from Applied Biosystems, Zwijndrecht, The Netherlands.

Techniques: Western Blot, Staining, Purification, Positive Control, Expressing