Journal: Journal of Clinical & Translational Endocrinology

Article Title: Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animal model

doi: 10.1016/j.jcte.2014.09.005

Figure Lengend Snippet: Screening of thyroid function to confirm hypothyroid status. ELISA were performed to measure T3 (A) and T4 (B) concentrations ( N = 6/group). (C) Detection of Deiodinase type 1 (DIO1) in uterine tissues obtained from control, hypothyroid and levothyroxine (T4)-treated non-pregnant rats. (D) Western blot quantification. This figure is representative of 5 identical experiments. * p < 0.05.

Article Snippet: Using a primary antibody raised against DIO1 (Santa Cruz, CA), an immunoreactive band of 78 kDa was consistently detected in all tested cytosolic samples while a 42 kDa band was detected using β-actin antibody ( C).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Scientific Reports

Article Title: A thyroid hormone network exists in synovial fibroblasts of rheumatoid arthritis and osteoarthritis patients

doi: 10.1038/s41598-019-49743-4

Figure Lengend Snippet: Synovial cells positive for deiodinase 1 (DIO1), the thyroid hormone transporter MCT8 (solute carrier family 16 member 2), thyroid hormone receptor alpha (TRα), and thyroid hormone receptor beta (TRβ). Scale bar = 60 µm. Abbreviations: Co., control staining; OA, osteoarthritis; RA, rheumatoid arthritis.

Article Snippet: Then, 6–8 μm sections were stained with a panel of antibodies directed against DIO1 (NPB1-19706, Novus Biologicals, via R&D Systems), DIO2 (ab135711, abcam, Cambridge, UK), DIO3 (NBP 1-05767, Novus Biologicals, via R&D Systems), MCT8 (thyroid hormone transporter, ab104689, abcam), TRα (ab42565, abcam), TRβ (ab5622, abcam), and trace amine associated receptor 1 (TA1, ab65633, abcam).

Techniques: Control, Staining

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 1. Low selenoenzyme type I deiodinase (DIO1) levels in high-grade serous ovarian cancer (HGSOC) cell lines and xenografts. CHO-K1 (normal ovaries), FT282, FT109, ES-2, and Kuramochi cells were analyzed for the DIO1 by (A) flow cytometry and (B) Western blots. A representative analysis of three experimental repeats is presented. b-tubulin was used for protein loading. Average DIO1 protein expression SEM is presented. *P < 0.05 and **P < 0.05 by ANOVA for multiple comparisons. (C) Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining for DIO1 and KI67 in formalin-fixed paraffin-embedded (FFPE) tumors from three mice. Scale bar, 20 lm. Matched isotype control is presented in Fig. S2A. 209 objective, Olympus microscopy (Olympus, Tokyo, Japan).

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Cytometry, Western Blot, Expressing, Immunohistochemistry, Control, Microscopy

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 2. Selenoenzyme type I deiodinase (DIO1) correlates with overall survival in high-grade serous ovarian cancer (HGSOC) patients. DIO1 expression (high vs. low) was analyzed in (A) HGSOC patients or (B) patients treated with carboplatin/taxol, who underwent optimal (no residual tumor) or suboptimal debulking surgery. Data were retrieved from The Cancer Genome Atlas (TCGA) using the Kaplan–Meier Plotter tool (http://kmplot.com). HR, hazard ratio. (C) Boxplots of DIO1 expression in responders vs. non-responders, after therapy comple- tion using the ROC Plotter (https://rocplot.com/custom-data/index). *P value < 0.05 following Mann–Whitney test. (D) DIO1 immunohisto- chemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) sections from human ovarian tumors (patients 1–10). Scale bar, 50 lm. Hematoxylin and eosin (H&E) and p53 staining were used to define tumor regions. p53 deleted cases (marked by an asterisk) underwent p16 staining. For each image, an enlarged inset of DIO1 expression is shown in the top right corner. Negative background stain was also confirmed using a matched isotype control antibody (Fig. S2B). 109 objective, Olympus microscopy (Olympus, Tokyo, Japan).

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Expressing, MANN-WHITNEY, Immunohistochemistry, Staining, Control, Microscopy

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 3. Selenoenzyme type I deiodinase (DIO1) expression in normal ovary, fallopian tubes, and throughout high-grade serous ovarian cancer (HGSOC) evolution. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) analyses of p53, KI67, PAX8, and DIO1 in representative formalin-fixed paraffin-embedded (FFPE) tissues of (A) normal ovary and fallopian tubes and (B) serous tubal in-situ carcinoma (STIC), primary and colon metastasis from a representative HGSOC patient (patient #7). Scale bar, 50 lm. 109 objective, Olympus microscopy (Olympus, Tokyo, Japan).

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Expressing, Immunohistochemistry, In Situ, Microscopy

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 4. Selenoenzyme type I deiodinase (DIO1) overexpression inhibits high-grade serous ovarian cancer (HGSOC) proliferation. ES-2 cells transiently transfected with empty PCDNA3 or DIO1-PCDNA3 expression vectors were analyzed after 3 days for (A) DIO1 protein levels in duplicates from control and DIO1 expression vectors are shown by Western blot (WB). b-tubulin was used for protein loading. Average DIO1 protein expression SEM is shown in the lower panel. (B) Light microscopy (X10 images, scale bar, 100 lm). Olympus microscopy images, using Cell^A software imaging. (C) Absolute cell counts, FC. (D) Annexin-PI histograms, FC. (E) Quantification of surviving cell population (Annexin/PI) and apoptotic cell population (Annexin+/PI+). Results (average SEM) were repeated twice, in triplicates. *P < 0.05 using Student’s unpaired t test.

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Over Expression, Transfection, Expressing, Control, Western Blot, Light Microscopy, Microscopy, Software, Imaging

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 5. Selenoenzyme type I deiodinase (DIO1) silencing induces proliferation and alters protein expression in ovarian cancer. Analyses of DIO1 level in knockdown (KD) versus control ES-2 cells for (A) mRNA transcription (RQ-PCR). (B) Light microscopy of 1 9 105 control and DIO1-KD cells incubated in 24-well plates for 96 h (910 images, scale bar, 500 lm). Olympus microscopy images, using Cell^A software imaging. Isolated clones from DIO1-KD versus control cells were analyzed for (C) DIO1 protein level by Western blot. (D) Cell number, by flow cytometry. (E) pERK, p21, HK1, GAPDH, PKM2, PDHA1, ATP5A, and Trip11 levels by Western blot. b-tubulin was used for protein loading. (F) Proteomics analyses of differentially expressed proteins in DIO1-KD high-grade serous ovarian cancer (HGSOC). Interaction network, divided into three clusters, using STRING v. 10.5 (https://string-db.org), with default settings (minimum required interaction score: medium confidence 0.4). (G) Pie chart using PANTHER (http://pantherdb.org). Cellular and metabolic processes are shown. (H) Proteomaps using the bionic visualization tool (BionicVis). (I) Proteomics expression analyses for a collection of ATP5 synthase complex subunits in con- trol and DIO1-KD cells. Results (average SEM) are shown as % of control. Results were repeated twice in triplicates. *P < 0.05. **P < 0.005 by Student’s unpaired t test.

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Expressing, Knockdown, Control, Light Microscopy, Incubation, Microscopy, Software, Imaging, Isolation, Clone Assay, Western Blot, Cytometry

Journal: Molecular oncology

Article Title: The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

doi: 10.1002/1878-0261.13612

Figure Lengend Snippet: Fig. 6. Selenoenzyme type I deiodinase (DIO1) inhibition promotes ovarian cancer cell growth. ES-2 cells were seeded (1000 cells/96 wells), treated with PTU at increasing concentrations, and analyzed after 96 h for (A) cell density by light microscopy (910 images, scale bar, 100 lm). Olympus microscopy images, using Cell^A software imaging. (B) Cell proliferation (CyQUANT, ELISA). (C) Cell viability (PrestoBlue, ELISA). Next, normal ovary cells (CHO-K1), HGSOC cells (ES-2), and scrambled and DIO1-KD ES-2 cells were treated with 10 lM propylthiouracil (PTU) and analyzed by (D) Light microscopy (910 images, scale bar, 100 lm). Olympus microscopy images, using Cell^A software imaging. (E) Absolute cell counts (flow cytometry). (F) Cell viability (PrestoBlue, ELISA). Results (average SEM) were repeated twice in triplicates. *P < 0.05. **P < 0.005 using ANOVA.

Article Snippet: Stable transfections with shRNA for DIO1 (sc-77146-SH, Santa Cruz Technologies, Dallas, TX, USA) were performed following the manufacturer’s instructions using shRNA plasmid transfection reagent (sc-108061, Santa Cruz Technologies).

Techniques: Inhibition, Light Microscopy, Microscopy, Software, Imaging, CyQUANT Assay, Enzyme-linked Immunosorbent Assay, Cytometry

Journal: Nature Communications

Article Title: Dido3-dependent HDAC6 targeting controls cilium size

doi: 10.1038/ncomms4500

Figure Lengend Snippet: RPE-1 cells were transduced with lentiviral particles expressing a non-targeting RNA interference (RNAi) or an RNAi directed against Dido . Transduced cells were then seeded on glass coverslips, grown until confluent and serum starved. Immunofluorescence labelling ( a ) and western blotting ( b ) with antibodies against human death inducer obliterator (Dido) showed efficient suppression. Measurement of cilium length ( c ) showed that Dido suppression resulted in significantly longer cilia (one-way analysis of variance). Horizontal dotted bar indicates average cilium size, error bars indicate standard error of the mean ( n =58). Note: Outliers (cilia with length>8 μm) were excluded for calculation of P -value. ( d – g ) Immunofluorescence with anti-ARL13B antibodies (green) confirmed Ac-tub (red) as an accurate marker of cilium size, even for extremely long cilia (right panel). ( e ) Magnification of boxed areas in d . Whereas ARL13B distributes evenly over the cilium in control cells, Dido RNAi causes redistribution to the cilium base and tip (arrows). Molecular weight of markers is indicated in kDa on the right of the western blots. Scale bars, 10 μm. Separate grey scale versions of the micrograph images are supplied in .

Article Snippet: For the knockdown of protein expression, small hairpin RNA against human (sc-35194-v) or mouse (sc-35195-v) Dido was delivered by lentiviral particles (Santa Cruz Biotechnology).

Techniques: Transduction, Expressing, Immunofluorescence, Western Blot, Marker, Control, Molecular Weight