Journal: Frontiers in Microbiology

Article Title: Intestinal Flora-Derived Kynurenic Acid Protects Against Intestinal Damage Caused by Candida albicans Infection via Activation of Aryl Hydrocarbon Receptor

doi: 10.3389/fmicb.2022.934786

Figure Lengend Snippet: KynA suppressed the MK2/p-MK2 signaling pathway by activating AHR. We investigated the regulation of TTP and expression changes of MK2 and P-MK2 induced by AHR activation at the cellular level. In Caco-2 cells, after treatment with KynA, expression of CYP1A1 increased significantly, as did expression of TTP also ( P < 0.05), while expression of MK2 decreased significantly ( P < 0.05). In the CA- Caco-2 cell model, the expression of proteins CYP1A1 and TTP decreased significantly, but the expression of p-MK2 increased ( P < 0.01). After the treatment of Caco-2 cells with KynA and the AHR agonist FICZ, the expression of CYP1A1 and TTP was significantly increased, but the expression of p-MK2 decreased significantly ( P < 0.01). Treatment with an AHR inhibitor (CH223191) led to a decrease in the expression of CYP1A1 and TTP, and an increase in the expression of p-MK2 ( P < 0.01). Statistical significance was evaluated using the Mann–Whitney U test. P -values < 0.05 (*) or < 0.01 (**) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid; FICZ, 6-Formylindolo[3,2-b]carbazole. P -values < 0.001 (***).

Article Snippet: Proteintech, United States); anti-AHR antibody (1:1,000, 67785-1-Ig; Proteintech, United States); anti-ZO-1 antibody (1:1,000, 21773-1-AP, Proteintech, United States); anti-GAPDH antibody (1:1,000, 60004-1-Ig, Proteintech, United States); anti-MK2 antibody (1:1,000, 13949-1-AP, Proteintech, United States); anti-CYP1A1 antibody (1:1,000, 13241-1-AP, Proteintech, United States); anti-TTP antibody (1:1,000, 12737-1-AP; Proteintech, United States); anti-MLCK antibody (1:1,000, 21642-1-AP; Proteintech, United States); and anti-pMLC antibody (1:2,000, CST-3671; Cell Signaling Technology, United States).

Techniques: Expressing, Activation Assay, MANN-WHITNEY, Infection

Journal: Nucleic Acids Research

Article Title: Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

doi: 10.1093/nar/gkr1302

Figure Lengend Snippet: TTP negatively regulates the levels of Lin28a in human cancer cells. ( A–D ) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (** P < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). ( E–I ) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (** P < 0.01; *** P < 0.001). ns, not significant. ( J ) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. ( K ) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.

Article Snippet: After deparaffinization, primary antibody anti-human TTP antibody (sc-14030, Santa Cruz Biotechnology) or anti-human Lin28a antibody (ab46020, Abcam) at a 1:100 dilution was applied for 2°h at room temperature.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Proliferation Assay, Control, Immunohistochemical staining, Staining

Journal: Nucleic Acids Research

Article Title: Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

doi: 10.1093/nar/gkr1302

Figure Lengend Snippet: TTP and Lin28a expression and clinicopathologic features of patients with ovarian adenocarcinoma

Article Snippet: After deparaffinization, primary antibody anti-human TTP antibody (sc-14030, Santa Cruz Biotechnology) or anti-human Lin28a antibody (ab46020, Abcam) at a 1:100 dilution was applied for 2°h at room temperature.

Techniques: Expressing, Significance Assay, Staining

Journal: Nucleic Acids Research

Article Title: Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

doi: 10.1093/nar/gkr1302

Figure Lengend Snippet: TTP enhances the decay of Lin28a mRNA through interaction with an ARE within the Lin28a mRNA 3′-UTR. ( A and B ) TTP destabilizes Lin28a mRNA. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The level of TTP protein was determined by western blot assays. (B) Expression of Lin28a mRNA in PA1 cells was determined by qRT-PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5.0 µg/ml actinomycin D. A one-phase model of exponential decay was used to derive the indicated mRNA decay curves. Results shown on the graph represent the means ± SD of three independent experiments (** P < 0.01; *** P < 0.001). ( C ) Inhibition of TTP by siRNA enhances Lin28a mRNA stability. HCT116 cells were transfected with siRNA against TTP-specific (TTP-siRNA) or scRNA for 24 h. Expression of Lin28a mRNA in HCT116 cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (* P < 0.05). ( D ) Lin28a mRNA half-life in PA1 cell with low TTP level is longer than that in AGS cells with high TTP level. Expression of Lin28a mRNA in PA1 and AGS cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (*** P < 0.001). ( E and F ) The first AUUUA pentamer (ARE1) within the Lin28a 3′-UTR is essential for the inhibitory effect of TTP. (E) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) and oligonucleotides (Oligo) derived from the Lin28a mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type (W) pentameric motif AUUUA; gray circles, mutated (M) motif AGCA. (F) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments (left panel) or oligonucleotides (right panel) derived from the Lin28a mRNA 3′-UTR as described in (E) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with the Frag-ARE1 luciferase construct alone or Oligo-ARE1W alone were set to 1.0. Results shown represent the means ± SD of three independent experiments (* P < 0.05; ** P < 0.01). ns, not significant. ( G and H ) TTP decreases the luciferase activity of luciferase reporter gene containing the Lin28b 3′-UTR. (G) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) derived from the Lin28b mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type pentameric motif AUUUA. (H) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments derived from the Lin28b mRNA 3′-UTR as described in (G) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with each Frag-ARE luciferase construct alone was set to 1.0. Results shown represent the means ± SD of three independent experiments (** P < 0.01; *** P < 0.001). ( I ) Ribonucleoprotein immunoprecipitation assay. PA1 cells were cotransfected with pcDNA6/V5-TTP and psiCHECK2 luciferase reporter constructs containing Lin28a Oligo-ARE1W. psiCHECK2 luciferase reporter construct containing mutant ARE1, Oligo-ARE1M was used as a negative control. At 24 h after transfection, the ribonucleoprotein complexes containing TTP were immunoprecipitated with protein G-agarose and anti-V5 or a control antibody. The luciferase mRNA in the immunoprecipitates was amplified by RT-PCR. The presence of TTP in the immunoprecipitates was detected by western blot with anti-V5 antibody. ( J and K ) RNA EMSA was performed by mixing cytoplasmic extracts containing 3.0 µg of total protein from pcDNA6/V5-TTP-transfected PA1 cells (J) or HCT116 cells (K) with 80 fmol biotinylated wild-type Oligo-ARE1W (WT) or mutant Oligo-ARE1M (MUT) probe. Anti-V5 (J), anti-TTP (K) or control antibody was added to the reaction mixtures. Position of the TTP containing bands (TTP) and super-shifted bands (SS) are indicated.

Article Snippet: After deparaffinization, primary antibody anti-human TTP antibody (sc-14030, Santa Cruz Biotechnology) or anti-human Lin28a antibody (ab46020, Abcam) at a 1:100 dilution was applied for 2°h at room temperature.

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Construct, Derivative Assay, Clone Assay, Plasmid Preparation, Activity Assay, Immunoprecipitation, Mutagenesis, Negative Control, Control, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

doi: 10.1093/nar/gkr1302

Figure Lengend Snippet: Overexpression of Lin28a attenuates the effects of TTP on let-7 b levels, CDC34 levels and PA1 cell growth. ( A–C ) Downregulation of Lin28a by siRNA increases let-7b levels but decreases CDC34 levels in PA1 cells. PA1 cells were transfected with Lin28a -specific (Lin28a-siRNA) or scRNA for 24 h. (A) The level of Lin28a was determined by qRT-PCR (top panel) and western blot assays (bottom panel). The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (*** P < 0.001). (B) The level of mature let-7b was measured by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (* P < 0.05). (C) The level of CDC34 was determined by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (*** P < 0.001). ( D–F ) Transfection of Lin28a cDNA without the 3′-UTR abolishes the effects of TTP on expression of let-7b , CDC34 and PA1 cell growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a for 24 h. (D) The levels of TTP and Lin28a were measured by semi-qRT-PCR (top panel) and western blot assays (bottom panel). (E) The level of let-7b was measured by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (* P < 0.05; ** P < 0.01). (F) Cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD ( n = 3) (** P < 0.01; *** P < 0.001).

Article Snippet: After deparaffinization, primary antibody anti-human TTP antibody (sc-14030, Santa Cruz Biotechnology) or anti-human Lin28a antibody (ab46020, Abcam) at a 1:100 dilution was applied for 2°h at room temperature.

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Proliferation Assay

Journal: Scientific Reports

Article Title: The Fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR Promotes the Growth of Hepatoma Cells by Upregulating HMGA2

doi: 10.1038/s41598-017-02311-0

Figure Lengend Snippet: HMGA2-sh-3p20 upregulates HMGA2 by blocking the TTP-mediated degradation of HMGA2 mRNA. ( A ) The relative expression of HMGA2-sh-3p20 was assessed by qRT-PCR in 35 pairs of clinical HCC tissues and corresponding peritumor tissues ( ***P < 0.001; Wilcoxon’s signed-rank test). ( B ) The correlation between HMGA2 mRNA levels and HMGA2-sh-3p20 levels was measured by qRT-PCR in 30 cases of clinical HCC tissues ( **P < 0.01, r = 0.586; Pearson’s correlation coefficient). ( C ) The luciferase activities of pGL3-HMGA2 were examined by luciferase reporter gene assays in HepG2 cells. ( D ) The expression of HMGA2 was assessed by qRT-PCR and Western blot analysis in Huh7 cells. ( E ) The diagram of TTP-mediated mRNA degradation. ( F ) The diagram of HMGA2-sh-3p20 antagonizes the interaction of TTP with non-hairpin within 3′UTR of HMGA2 mRNA. ( G ) TTP RIP-PCR of HMGA2 in HepG2 cells. ( H ) Effect of TTP on the expression of HMGA2 was measured by qRT-PCR and Western blot analysis in HepG2 cells. ( I ) Effect of HMGA2-sh-3p20 on the expression of TTP-mediated HMGA2 was assessed by qRT-PCR and Western blot analysis in HepG2 cells. The full length blots images are given as Supplementary Fig. . ( J ) TTP RIP-qPCR of HMGA2 in HepG2 cells transfected with HMGA2-sh-3p20. ( K ) Effect of HMGA2-sh-3p20 on the levels of HMGA2 mRNA in HepG2 cells transfected with si-TTP by qRT-PCR. ( L , M ) Effect of HMGA2-sh-3p20 on the half-life of HMGA2 mRNA in HepG2 cells ( L ) or HepG2 cells transfected with si-TTP ( M ) by qRT-PCR. Every experiment was repeated three times. Error bars represent s.d. (n = 3), **p < 0.01; ***p < 0.001 and not significant (NS), Student’s t test.

Article Snippet: The dilution of primary antibody is following: HMGA2 (1: 5000, Genetex), PTEN (1:800, Proteintech), TTP (1:1000, Proteintech), Drosha (1:1000, Proteintech), DGCR8 (1:800, Proteintech), Dicer (1:800, Proteintech), β-actin (1:2000, Abcam).

Techniques: Blocking Assay, Expressing, Quantitative RT-PCR, Luciferase, Western Blot, Transfection

Journal: Scientific Reports

Article Title: The Fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR Promotes the Growth of Hepatoma Cells by Upregulating HMGA2

doi: 10.1038/s41598-017-02311-0

Figure Lengend Snippet: A model shows that the fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR promotes the growth of hepatoma cells by upregulating HMGA2. Bioinformatics analysis shows that 3′UTR of HMGA2 mRNA contains the hairpin structure (HMGA2-sh). Drosha and DGCR8 cleave the HMGA2-sh from the 3′UTR of HMGA2 mRNA, and Dicer contributes to the generation of the HMGA2-sh-3p20 from the HMGA2-sh. Furthermore, HMGA2-sh-3p20 is able to increase the levels of HMGA2 by antagonizing TTP-mediated HMGA2 degradation, while it decreases PTEN by targeting 3′UTR of PTEN mRNA. In addition, the downregulated-PTEN is not able to depress the expression of HMGA2, leading to the upregulation of HMGA2. Functionally, HMGA2-sh-3p20-enhanced HMGA2 accelerates the growth of liver cancer cells.

Article Snippet: The dilution of primary antibody is following: HMGA2 (1: 5000, Genetex), PTEN (1:800, Proteintech), TTP (1:1000, Proteintech), Drosha (1:1000, Proteintech), DGCR8 (1:800, Proteintech), Dicer (1:800, Proteintech), β-actin (1:2000, Abcam).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2)

doi: 10.3390/ijms17071016

Figure Lengend Snippet: Expression of human α-tocopherol transfer protein (α-TTP) eukaryotic expression vector in Human Hepatoma cells (HepG2). ( A ) Result of quantitative real-time PCR; ( B ) Result of Western blot; ( C ) Result of Immunofluorescence (10×). pcDNA: pcDNA3.1-mycHisa empty vector; p-TTP: human α-TTP eukaryotic expression vector.

Article Snippet: The primary antibody was human α-TTP monoclonal (Abnova, Taiwan, Dilution ratio 1:1000) and the secondary antibody was goat anti-mouse (Dilution ratio 1:5000) (from Beijing B & M Biotech Co., Ltd., Beijing, China).

Techniques: Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2)

doi: 10.3390/ijms17071016

Figure Lengend Snippet: Expression levels of α-TTP related genes. SEC23A : Sec23 homolog A; RTP4 : receptor transporter protein 4; CLIC3 : chloride intracellular channel 3; CENPE : centromere protein E; KCNQ1 : potassium voltage-gated channel subfamily Q member 1; GOLGA4 : golgi autoantigen, golgin subfamily a, 4; BNIP3 : BCL2/adenovirus E1B 19 kDa interacting protein 3; CENPF : centromere protein F. Data are presented as mean ± standard error; the asterisk indicated significant difference between control and treatment group.

Article Snippet: The primary antibody was human α-TTP monoclonal (Abnova, Taiwan, Dilution ratio 1:1000) and the secondary antibody was goat anti-mouse (Dilution ratio 1:5000) (from Beijing B & M Biotech Co., Ltd., Beijing, China).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2)

doi: 10.3390/ijms17071016

Figure Lengend Snippet: Composition of the ligation reaction.

Article Snippet: The primary antibody was human α-TTP monoclonal (Abnova, Taiwan, Dilution ratio 1:1000) and the secondary antibody was goat anti-mouse (Dilution ratio 1:5000) (from Beijing B & M Biotech Co., Ltd., Beijing, China).

Techniques: Ligation, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2)

doi: 10.3390/ijms17071016

Figure Lengend Snippet: Details of primers used for quantitative real-time PCR.

Article Snippet: The primary antibody was human α-TTP monoclonal (Abnova, Taiwan, Dilution ratio 1:1000) and the secondary antibody was goat anti-mouse (Dilution ratio 1:5000) (from Beijing B & M Biotech Co., Ltd., Beijing, China).

Techniques: Sequencing