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: 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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

doi: 10.1073/pnas.2310409121

Figure Lengend Snippet: Fig. 1. Pathogenic mutant in familial ovarian immature teratoma. (A) Cases from two pedigree of OIT and their corresponding genotypes of BMP15. (B) Pathological diagnosis of corresponding patients. Blue dash-circled areas and arrowheads indicate two to three germ layer-derived tissues in the tumor tissue; neural components and neuroglia in the ectoderm; and the neuroepithelium is connected into pieces in a daisy doughnut or irregular glandular shape. (C) Sanger sequencing results of corresponding patients. (D) Conservation of Arg 88 across various species. (E) Hydrophilicity of Arg 88 across various species. (F) Predicted structural change of c.C262T (MT) mutated BMP15.

Article Snippet: The slides were incubated with the primary antibody at 4 °C overnight for BMP15 (rabbit polyclonal antibody to mouse, Proteintech: Catalog Number18982- 1- AP) Do w nl oa de d fr om h ttp s: //w w w .p na s. or g by U ni v Fe de ra l d a Pa ra ib a on A ug us t 3 0, 2 02 4 fr om I P ad dr es s 15 0.

Techniques: Mutagenesis, Biomarker Discovery, Derivative Assay, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

doi: 10.1073/pnas.2310409121

Figure Lengend Snippet: Fig. 3. C262T mutant significantly stimulates parthenogenetic activation of oocytes. Oocytes were collected from genetically engineered mice harboring the orthologous of BMP15 wild type or C262T variants. Superovulation was induced at 4 wk of age mice. Ten ovaries were assessed from each genotype. The parthenogenetic activation in oocyte was assessed by morphological changes (panel A–C) or histological stain (panel D and E). (A) Two pronucleus were seen in oocyte with spontaneous parthenogenetic activation (yellow arrowhead); (B) two pronucleus and two polar bodies were seen in oocyte with spontaneous parthenogenetic activation (yellow and blue arrowheads, respectively); (C) Two-cell (green arrowheads) was seen with spontaneous parthenogenetic activation; (D) The oocytes appear to have two pronucleus in the HE section (yellow arrowhead); (E) Two polar bodies and one pronucleus appear in the oocyte in the HE section (yellow and blue arrowheads, respectively); (F) Two-cell (green arrowheads) appeared in HE sections; (G) The percentage of parthenogenetically activated oocytes in different BMP15 variants was counted under the Leica DMI3000B inverted biological microscope.

Article Snippet: The slides were incubated with the primary antibody at 4 °C overnight for BMP15 (rabbit polyclonal antibody to mouse, Proteintech: Catalog Number18982- 1- AP) Do w nl oa de d fr om h ttp s: //w w w .p na s. or g by U ni v Fe de ra l d a Pa ra ib a on A ug us t 3 0, 2 02 4 fr om I P ad dr es s 15 0.

Techniques: Mutagenesis, Activation Assay, Staining, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

doi: 10.1073/pnas.2310409121

Figure Lengend Snippet: Fig. 2. C262T mutant affects the protein expression of BMP15. (A) Wild type and C262T mutated BMP15 were overexpressed in HEK293 cells. Amounts of secreted BMP15 (s-BMP15) were compared by western blot. Signal intensity was semi-quantified using ImageJ with the arbitrary unit (P < 0.05). (B) The general BMP15 protein level in a healthy ovarian, BMP15 was highly expressed in both granulosa cells (red arrow) and oocytes (blue arrow) of healthy ovaries; (C) Wild-type BMP15 in sporadic ovarian immature teratoma, BMP15 is highly expressed in the follicular epithelium of tumors (black arrow). (D) C262T variant in familial ovarian immature teratoma. The expression level of the mature BMP15 protein was significantly decreased in immature teratomas carrying the C262T mutation (blue arrow).

Article Snippet: The slides were incubated with the primary antibody at 4 °C overnight for BMP15 (rabbit polyclonal antibody to mouse, Proteintech: Catalog Number18982- 1- AP) Do w nl oa de d fr om h ttp s: //w w w .p na s. or g by U ni v Fe de ra l d a Pa ra ib a on A ug us t 3 0, 2 02 4 fr om I P ad dr es s 15 0.

Techniques: Mutagenesis, Expressing, Western Blot, Variant Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

doi: 10.1073/pnas.2310409121

Figure Lengend Snippet: Fig. 4. H-Ras activated MAPK stimulate spontaneous activation of the oocytes and overgrown of the ovary. (A) Venn diagram showing the enriching strategy of oocytes specific RNA sequencing results. (B) Heat map showing the distinct diverse pattern of differential expressed genes in oocytes with wild-type or mutated BMP15. (C) H-Ras and MEK1 RNA levels in CC, CT, or TT type oocytes. (D) IHC stain showing the BMP15, H-Ras, MEK1, and p-MEK1 protein levels in CC, CT, or TT type ovarian tissues at week 4. (E) (i) Hematoxylin and eosin stain of ovarian tissues at the longest latitude section; blue arrowheads show follicles; (ii) the actual ovary size of CC and CT type at month 6; (iii) average counts of ovarian follicles per slicing section at month 6. PF, primary follicle; SF, secondary follicle; MF, mature follicle; AF, atretic follicle; *** indicates P-value < 0.001. (F) An ovarian teratoma in the CT type ovary. Blue arrowhead shows a digestive tract structure composed of layers of epithelial cells. (G) IHC stain showing the BMP15, H-Ras, MEK1, and p-MEK1 protein levels in a CC type healthy ovary and the CT type ovary with the teratoma at month 6.

Article Snippet: The slides were incubated with the primary antibody at 4 °C overnight for BMP15 (rabbit polyclonal antibody to mouse, Proteintech: Catalog Number18982- 1- AP) Do w nl oa de d fr om h ttp s: //w w w .p na s. or g by U ni v Fe de ra l d a Pa ra ib a on A ug us t 3 0, 2 02 4 fr om I P ad dr es s 15 0.

Techniques: Activation Assay, RNA Sequencing, Staining, H&E Stain