Journal: International Journal of Molecular Sciences

Article Title: Selective Cytotoxicity of Complexes with N,N,N-Donor Dipodal Ligand in Tumor Cells

doi: 10.3390/ijms22041802

Figure Lengend Snippet: The effect of complexes 1, 2 and 3 and their salts on the expression of genes controlling angiogenesis and invasive/metastatic phenotype of tumour cells. The expression of mRNA for vascular endothelial growth factor a (VEGFa) (A), vimentin (Vim) (B), matrix metalloproteinase-2 (MMP-2) (C) and matrix metalloproteinase-9 (MMP-9) (D) in Hep G2 cells was analysed with Real-Time PCR (qPCR). The cells (2 × 10 4 /well) were exposed to 50 μM/L of each compound (or appropriate comparator salt) for 24 h. The data were normalized against reference gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript; the 2 −ΔΔCt method was used for determination of mRNA level; * p < 0.05 and ** p < 0.01 vs. untreated control). The real-time PCR data were presented as arithmetic mean values and standard deviation (M; ±SD), n = 3.

Article Snippet: The following Taq-Man human probes (Applied Biosystems) were used for qPCR reaction: VEGFA (Hs00173626_m1), VIM (001855284_m1), MMP-2 (Hs00234422_m1), MMP-9 (Hs00234579_m1) and GAPDH (Hs99999905_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Selective Cytotoxicity of Complexes with N,N,N-Donor Dipodal Ligand in Tumor Cells

doi: 10.3390/ijms22041802

Figure Lengend Snippet: The effect of complexes 1, 2 and 3 and their salts on the expression of genes controlling angiogenesis and invasive/metastatic phenotype of tumour cells. The expression of mRNA for vascular endothelial growth factor a (VEGFa) (A), vimentin (Vim) (B), matrix metalloproteinase-2 (MMP-2) (C) and matrix metalloproteinase-9 (MMP-9) (D) in Hep G2 cells was analysed with Real-Time PCR (qPCR). The cells (2 × 10 4 /well) were exposed to 50 μM/L of each compound (or appropriate comparator salt) for 24 h. The data were normalized against reference gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript; the 2 −ΔΔCt method was used for determination of mRNA level; * p < 0.05 and ** p < 0.01 vs. untreated control). The real-time PCR data were presented as arithmetic mean values and standard deviation (M; ±SD), n = 3.

Article Snippet: The following Taq-Man human probes (Applied Biosystems) were used for qPCR reaction: VEGFA (Hs00173626_m1), VIM (001855284_m1), MMP-2 (Hs00234422_m1), MMP-9 (Hs00234579_m1) and GAPDH (Hs99999905_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: BMC Cancer

Article Title: Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

doi: 10.1186/s12885-017-3418-y

Figure Lengend Snippet: Pharmacological inhibitors of MMP-2 and MMP-9 downregulate MMP2 and MMP9 mRNA. a The following MMPs were examined at the transcriptional level: MMP2 , MMP7 , MMP9 , and MMP14 . Y79 ( left ) and Weri-1 ( right ) cells were harvested for RNA isolation and cDNA synthesis. Material was pre-amplified using the TaqMan® PreAmp Master Mix with the respective primers. qPCR was done and results show mRNA expression relative to HPRT1 as endogenous control. Bar graphs indicate results ±SD; n = 3 biological replicates in triplicates. Y79 Rb cells express MMP2 , MMP9 and MMP14 ; Weri-1 expressed MMP2 and MMP9 . b - c Y79 ( b ) and Weri-1 ( c ) cells were treated with MMP-2 and MMP-9 inhibitors overnight. RNA and cDNA was extracted as in a showing that the inhibitors act at the transcriptional level. Bar graphs indicate fold change ±SD; n = 3. Standard deviation obtained from the biological replicates. d Knockdown of MMP2 and MMP9 by RNA interference shows on-target effects. Downregulation of MMP2 and MMP9 after siRNA compared to scramble samples. qPCR done as in a . e - f Reduction of MMP-2 and MMP-9 protein in Rb cells treated with MMPI ( e ) and siRNA ( f ); * p < 0.05, ** p < 0.005. Western blot bar graphs indicate results ±SEM ratio of target protein to β-actin; n = 3. g - h ELISA analyses of MMP-2 and MMP-9 protein of whole cell lysates after treatment with MMPI ( g ) or siRNA ( h ); * p < 0.05, ** p < 0.005. i - j , E2F regulates MMP expression in Y79 cells. Y79 cells treated with MMPI ( i ) or with siRNA ( j ) were assessed by Wb analysis for E2F. Western blot bar graphs indicate results ±SEM ratio of target protein to β-actin; n = 3; ** p < 0.005

Article Snippet: We used the following Human TaqMan® Gene Expression Assays: HPRT1 (Hs02800695_m1), MMP2 (Hs01548727_m1), MMP7 (Hs01042796_m1), MMP9 (Hs00234579_m1), MMP14 (Hs01037003_g1) all from Life Technologies (Grand Island, NY).

Techniques: Isolation, cDNA Synthesis, Amplification, Expressing, Control, Standard Deviation, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Antipsychotic Drug Aripiprazole Protects Liver Cells from Oxidative Stress

doi: 10.3390/ijms23158292

Figure Lengend Snippet: Mitochondrial UPR. Logarithmic fold-change in mRNA expression of long-term treated Fao of ( a ) Hspd1/Hsp60 ( n = 4), ( c ) Txn2 ( n = 4), ( d ) Clpp ( n = 3), and ( e ) Ymel1l ( n = 3); protein amounts long-term treated Fao standardized to total protein acquired from Coomassie-stained gel of ( b ) HSP60 with the corresponding blot ( n = 4). Data are presented as the mean ± standard deviation (SD) and analyzed with one-way ANOVA followed by Dunnett’s test, comparing all samples to the baseline control with no added H 2 O 2 (*); samples with 1.5 mM H 2 O 2 added are also compared to their untreated control at 1.5 mM H 2 O 2 ( # ). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n : number of biological replicates.

Article Snippet: The following TaqMan probes labeled with the FAM dye (Thermo Fisher Scientific) were used: the reference gene Rn18s (Rn03928990_g1), Nrf2/Nfe2l2 (Rn00582415_m1), Srxn1 (Rn04337926_g1), Sirt1 (Rn01428096_m1), Foxo3α (Rn01441087_m1), Bip/Hspa5 (Rn00565250_m1), Chop/Ddit3 (Rn00492098_g1), p53 (Rn00755717_m1), p21 (Rn00589996_m1), Gadd45α (Rn00577049_m1), Gadd45 β (Rn01452530_g1), Gadd45 γ (Rn01352550_g1), Icam1 (Rn00564227_m1), Hspd1 (Rn01441529_g1), Txn2 (Rn00584162_g1), Clpp (Rn01527475_m1), Ymel1l (Rn00586650_m1).

Techniques: Expressing, Staining, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Antipsychotic Drug Aripiprazole Protects Liver Cells from Oxidative Stress

doi: 10.3390/ijms23158292

Figure Lengend Snippet: Summary statistical results with probability values (P). Gray box: no statistical significance ( p > 0.05); green box : gene upregulation, more protein or higher enzyme activity; red box : gene downregulation, less protein, decreased cell viability or enzyme activities; white box: results are not presented/acquired due to reduced cell viability. Prot.: protein, exp.: expression, erUPR: endoplasmic reticulum unfolded protein response, mtUPR: mitochondrial unfolded protein response.

Article Snippet: The following TaqMan probes labeled with the FAM dye (Thermo Fisher Scientific) were used: the reference gene Rn18s (Rn03928990_g1), Nrf2/Nfe2l2 (Rn00582415_m1), Srxn1 (Rn04337926_g1), Sirt1 (Rn01428096_m1), Foxo3α (Rn01441087_m1), Bip/Hspa5 (Rn00565250_m1), Chop/Ddit3 (Rn00492098_g1), p53 (Rn00755717_m1), p21 (Rn00589996_m1), Gadd45α (Rn00577049_m1), Gadd45 β (Rn01452530_g1), Gadd45 γ (Rn01352550_g1), Icam1 (Rn00564227_m1), Hspd1 (Rn01441529_g1), Txn2 (Rn00584162_g1), Clpp (Rn01527475_m1), Ymel1l (Rn00586650_m1).

Techniques: Activity Assay, Expressing, Antioxidant Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Regulatory Roles of Chemerin-Chemokine-Like Receptor 1 Axis in Placental Development and Vascular Remodeling During Early Pregnancy

doi: 10.3389/fcell.2022.883636

Figure Lengend Snippet: Cmklr1 knockout is associated with increased dNK cells. (A) The amount and distribution of dNK cells of wildtype and Cmklr1 −/− were investigated by immunohistochemically staining with antibodies against DBA Lectin in different magnifications. Scale bar = 200 μm. Quantification of the number of dNK cells. (B) The concentration of IL-15 in serum of wildtype and Cmklr1 −/− at GD 12 was measured by ELISA. Total RNA samples from wildtype and Cmklr1 −/− placentas at GD12 were subjected to qRT-PCR. The middle placenta section from each litter (four litters from four mothers per group) was used for analysis. The expression of (C) IL-8, (D) MMP2, (E) MMP9, (F) IL-10, (G) TNF-α and (H) IL-6 were examined. The results were represented as mean ± SD, n = 4. * p < 0.05 and ** p < 0.01. IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; MMP: Matrix Metallopeptidase; TIMP: Tissue inhibitor of metalloproteinases. qRT-PCR: quantitative reverse-transcription polymerase chain reaction; SD: standard deviation. TPBPA: trophoblast-specific protein alpha.

Article Snippet: The TaqManTM Gene Expression Assay primers were purchased from Life Technologies, including mouse Interleukin (IL)-6 (Mm00446191_m1); IL-8 (Mm04208136_m1); IL-10 (Mm01288386_m1); Tumor necrosis factor-alpha (TNF-α) (Mm00443258_m1); Matrix metalloproteinase-2 (MMP)2 (Mm01253624_m1); MMP9 (Mm00600157_g1); Human Glial Cells Missing-1 (GCM1) (Hs00961601_m1); syncytin-1 (Hs00835189_CE); Caudal-type homeobox gene 2 (CDX2) (Hs01078080_m1); Human leukocyte antigen G (HLA-G) (Hs00365950_g1); and CK7 (Hs00559840_m1).

Techniques: Knock-Out, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: BMC Cancer

Article Title: Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

doi: 10.1186/s12885-017-3418-y

Figure Lengend Snippet: Pharmacological inhibitors of MMP-2 and MMP-9 downregulate MMP2 and MMP9 mRNA. a The following MMPs were examined at the transcriptional level: MMP2 , MMP7 , MMP9 , and MMP14 . Y79 ( left ) and Weri-1 ( right ) cells were harvested for RNA isolation and cDNA synthesis. Material was pre-amplified using the TaqMan® PreAmp Master Mix with the respective primers. qPCR was done and results show mRNA expression relative to HPRT1 as endogenous control. Bar graphs indicate results ±SD; n = 3 biological replicates in triplicates. Y79 Rb cells express MMP2 , MMP9 and MMP14 ; Weri-1 expressed MMP2 and MMP9 . b - c Y79 ( b ) and Weri-1 ( c ) cells were treated with MMP-2 and MMP-9 inhibitors overnight. RNA and cDNA was extracted as in a showing that the inhibitors act at the transcriptional level. Bar graphs indicate fold change ±SD; n = 3. Standard deviation obtained from the biological replicates. d Knockdown of MMP2 and MMP9 by RNA interference shows on-target effects. Downregulation of MMP2 and MMP9 after siRNA compared to scramble samples. qPCR done as in a . e - f Reduction of MMP-2 and MMP-9 protein in Rb cells treated with MMPI ( e ) and siRNA ( f ); * p < 0.05, ** p < 0.005. Western blot bar graphs indicate results ±SEM ratio of target protein to β-actin; n = 3. g - h ELISA analyses of MMP-2 and MMP-9 protein of whole cell lysates after treatment with MMPI ( g ) or siRNA ( h ); * p < 0.05, ** p < 0.005. i - j , E2F regulates MMP expression in Y79 cells. Y79 cells treated with MMPI ( i ) or with siRNA ( j ) were assessed by Wb analysis for E2F. Western blot bar graphs indicate results ±SEM ratio of target protein to β-actin; n = 3; ** p < 0.005

Article Snippet: We used the following Human TaqMan® Gene Expression Assays: HPRT1 (Hs02800695_m1), MMP2 (Hs01548727_m1), MMP7 (Hs01042796_m1), MMP9 (Hs00234579_m1), MMP14 (Hs01037003_g1) all from Life Technologies (Grand Island, NY).

Techniques: Isolation, cDNA Synthesis, Amplification, Expressing, Control, Standard Deviation, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Myeloid differentiation primary response gene (MyD) 88 signalling is not essential for intestinal fibrosis development

doi: 10.1038/s41598-017-17755-7

Figure Lengend Snippet: MyD88 deficiency reduces intestinal inflammation but not the development of intestinal fibrosis. ( A ) Significant increase of Mmp9 mRNA expression in grafts isolated from GFP-Tg recipients at day 14 in comparison to freshly isolated intestine. Mmp9 mRNA expression in grafts from MyD88 −/− recipients remained unchanged over time (p < 0.05 (*), error bars = SEM, n = 3 each column). ( B ) Timp1 mRNA expression remained identical over time in both grafts from GFP-Tg and MyD88 −/− recipients (n = 3 each column). ( C ) IHC revealed an increase in Ly-6G + neutrophils in grafts extracted from both GFP-Tg and MyD88 −/− donor animals compared to freshly isolated small intestine. The number of Ly-6G positive cells (yellow crosses) was significantly increased in grafts extracted from GFP-Tg (p < 0.05 (*), Kruskal-Wallis One Way Analysis of Variance on Ranks, All Pairwise Multiple Comparison Procedures, Dunn’s Method). The number of Ly-6G positive neutrophils was calculated from at least four places in representative areas at 20-fold magnification. Mean value und standard deviation is shown. n = 4 – 7 as indicated.

Article Snippet: Quantitative mRNA analysis by real-time PCR was performed using Taqman ® gene Expression Assays for mouse collagen type I alpha 1 (Col1a1) Mm00801666_g1, TGF-β1 Mm01178820_m1, MMP-9 Mm00442991_m1, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) Mm00441818_m1, hypoxia inducible factor 1 alpha subunit (HIF-1α) Mm01283760_m1 and GAPDH # 4352339E TaqMan® Gene Expression Assays.

Techniques: Expressing, Isolation, Comparison, Standard Deviation

Journal: PLoS Genetics

Article Title: A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1

doi: 10.1371/journal.pgen.1007532

Figure Lengend Snippet: (A) Manhattan plot of GWAS results in the isolated BA cohort. X-axis: genomic coordinates of tested SNPs. Y-axis: significance level on a -log 10 scale. The genome-level significance threshold is indicated by the red horizontal line ( P = 5 × 10 −8 ) and the suggestive significance threshold is indicated by the blue horizontal line ( P = 1 × 10 −5 ). (B) Regional association plot of 2p16.1 after meta-analysis. P -values (left Y-axis) obtained from additive frequentist association test on genotyped and imputed SNPs. The recombination rate (right Y-axis) is calculated from the 1000 Genomes Phase 3 European ancestry dataset. The top BA-associated SNP is imputed SNP rs6761893 (purple circle), located in the fifth intron of the gene EFEMP1 . The colors refer to r-square correlation of each SNP to rs6761893 based on the 1000 Genomes Phase 3 European ancestry dataset. The circles represent the genotyped markers and squares represent imputed markers.

Article Snippet: Human EFEMP1 primer and probe set labeled with FAM (Hs00244575_m1) and human TBP primer and probe set labeled with VIC (Hs00427620_m1) were used.

Techniques: Isolation

Journal: PLoS Genetics

Article Title: A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1

doi: 10.1371/journal.pgen.1007532

Figure Lengend Snippet: (A) ddPCR showing relative expression of EFEMP1 transcripts in human liver specimens. Fold change of EFEMP1 was determined by normalizing to the reference gene, TBP . Error bars indicate standard deviation. (B) Relative expression of Efemp1 transcripts in six cell populations from normal rat livers. Rat gene Rsp12 was used as reference gene. Error bars indicate standard deviation of technical triplicates (cholangiocytes, portal fibroblasts, hepatocytes, Kupffer cells, and sinusoidal endothelial cells) or technical duplicates (hepatic stellate cells).

Article Snippet: Human EFEMP1 primer and probe set labeled with FAM (Hs00244575_m1) and human TBP primer and probe set labeled with VIC (Hs00427620_m1) were used.

Techniques: Expressing, Standard Deviation

Journal: PLoS Genetics

Article Title: A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1

doi: 10.1371/journal.pgen.1007532

Figure Lengend Snippet: (A) EFEMP1 is expressed in SMA positive smooth muscle cells, but not (B) CK19 positive intrahepatic cholangiocytes in control liver. EFEMP1 is expressed in both smooth muscle cells and intrahepatic cholangiocytes in (C, D) BA, (E, F) TPN, and (G, H) ARPKD liver. Scale bar = 25 μm. (A-F) were taken at a lower magnification and correspond to the scale bar in (F). (G, H) were taken at a higher magnification and correspond to the scale bar in (H). Arrowheads indicate vascular smooth muscle cells and arrows indicate cholangiocytes.

Article Snippet: Human EFEMP1 primer and probe set labeled with FAM (Hs00244575_m1) and human TBP primer and probe set labeled with VIC (Hs00427620_m1) were used.

Techniques: Control

Journal: PLoS Genetics

Article Title: A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1

doi: 10.1371/journal.pgen.1007532

Figure Lengend Snippet: EFEMP1 is expressed in both (A) SMA positive smooth muscle cells and (B) CK19 positive extrahepatic cholangiocytes in BA liver. Scale bar = 25 μm.

Article Snippet: Human EFEMP1 primer and probe set labeled with FAM (Hs00244575_m1) and human TBP primer and probe set labeled with VIC (Hs00427620_m1) were used.

Techniques:

Journal: Scientific Reports

Article Title: Myeloid differentiation primary response gene (MyD) 88 signalling is not essential for intestinal fibrosis development

doi: 10.1038/s41598-017-17755-7

Figure Lengend Snippet: MyD88 deficiency reduces intestinal inflammation but not the development of intestinal fibrosis. ( A ) Significant increase of Mmp9 mRNA expression in grafts isolated from GFP-Tg recipients at day 14 in comparison to freshly isolated intestine. Mmp9 mRNA expression in grafts from MyD88 −/− recipients remained unchanged over time (p < 0.05 (*), error bars = SEM, n = 3 each column). ( B ) Timp1 mRNA expression remained identical over time in both grafts from GFP-Tg and MyD88 −/− recipients (n = 3 each column). ( C ) IHC revealed an increase in Ly-6G + neutrophils in grafts extracted from both GFP-Tg and MyD88 −/− donor animals compared to freshly isolated small intestine. The number of Ly-6G positive cells (yellow crosses) was significantly increased in grafts extracted from GFP-Tg (p < 0.05 (*), Kruskal-Wallis One Way Analysis of Variance on Ranks, All Pairwise Multiple Comparison Procedures, Dunn’s Method). The number of Ly-6G positive neutrophils was calculated from at least four places in representative areas at 20-fold magnification. Mean value und standard deviation is shown. n = 4 – 7 as indicated.

Article Snippet: Quantitative mRNA analysis by real-time PCR was performed using Taqman ® gene Expression Assays for mouse collagen type I alpha 1 (Col1a1) Mm00801666_g1, TGF-β1 Mm01178820_m1, MMP-9 Mm00442991_m1, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) Mm00441818_m1, hypoxia inducible factor 1 alpha subunit (HIF-1α) Mm01283760_m1 and GAPDH # 4352339E TaqMan® Gene Expression Assays.

Techniques: Expressing, Isolation, Comparison, Standard Deviation