Immunohistochemical detection of LCN2 and GPC3.

<p>(A) LCN2 staining in normal thyroid. (B) LCN2 staining in PTC. (C) GPC3 staining in normal thyroid. (D) GPC3 staining in PTC. (E) Proportions of positive and negative samples in each tissue group.</p

Expression of <i>PAX8</i> Target Genes in Papillary Thyroid Carcinoma - Fig 2

<p><b>Putative PAX8 target genes whose mRNA levels were most strongly correlated with those of <i>PAX8</i> in PTCs</b> Levels of mRNA for <i>ATP1B1</i> (A) and <i>KCNIP3</i> (B) are plotted against PAX8 mRNA levels measured in 31 CT-PTCs (•) and 5 FV-PTCs (○). R and p values were calculated with the Spearman rank correlation test.</p

Expression of putative PAX8 target genes in the 36 PTCs.

<p>Expression of putative PAX8 target genes in the 36 PTCs.</p

PCA of PAX8 target genes displaying marked PTC-related dysregulation.

<p>Analysis was performed with data for the four <i>PAX8</i> target genes (<i>LCN2</i>, <i>GPC3</i>, <i>KCNIP3</i>, <i>SCD1</i>) that were most markedly dysregulated in PTCs (red diamonds) compared with normal thyroid (NT) tissues (green diamonds). (A) Our dataset (36 PTC and 18 NTs). (B) TCGA dataset (486 PTC and 59 NTs). PCA was performed with the built-in prcomp function in R software.</p

Expression of putative PAX8 target genes in the 36 PTCs stratified by ATA risk and <i>BRAF</i> mutational status.

<p>Expression of putative PAX8 target genes in the 36 PTCs stratified by ATA risk and <i>BRAF</i> mutational status.</p

<i>PAX8</i> expression in normal and tumor thyroid tissues of PTC patients.

<p>(A) Mean (SD) <i>PAX8</i> mRNA levels found in normal thyroid tissuesamples (n = 18) and PTCs (n = 36), assessed by qPCR. (B-D) Representative images of immunohistochemical staining for PAX8 protein levels in (B) normal thyroid tissues; (C) PAX8-positive PTCs; and (D) PAX8-negative PTCs. (E) Mean H scores for immunohistochemical labeling of PAX8 protein in normal thyroid tissues (12 samples) and PTC (38 samples).</p

Supplemental materials and methods.

(DOCX)</p

MAB21L1 Arg51 and Phe52 substitution causes microphthalmia and aniridia.

A. Pedigrees are shown for the six families with MAB21L1 variants and bilateral microphthalmia and/or aniridia. The pedigrees are ordered by variant: c.152G>A p.(Arg51Gln) (orange shaded box), c.152G>T p.(Arg51Leu) (green shaded box), c.152G>C p.(Arg51Pro) (yellow shaded box) and c.155T>G p.(Arg52Cys) (pink shaded box). A key to the pedigree symbols is shown to the left (grey shaded box). B. A schematic of MAB21L1 represented as a linear bar and with the first and last amino acid residue numbered. The linear positions of all pathogenic variants are shown: The monoallelic variants in this study are detailed above (red text) and the published biallelic variants are detailed below (bracketed black text). C. Clinical images of individuals with MAB21L1 Arg51-related eye malformations. R, right eye; L, left eye. Family 511 all have profound aniridia, microcornea, choroidal coloboma (just visible in II:1’s L fundus photo) and optic disc anomalies. The progression of disease in II:1 over one decade is shown between with the upper and lower photos, with worsening of phthisis in the right and pannus in the left. An enlarged retroilluminated image of III:1’s L eye is shown highlighting near-total aniridia. Individual 1434 II:1, showing bilateral partial aniridia and microphthalmia, worse on the L. Abbreviations: dn, de novo. Nucleotide and amino acid numbering are based on GenBank NM_005584.5 and GenPept NP_005575.1, respectively.</p

Fig 2 -

A. Nucleotidyltransferase activity: Graph showing the absence of nucleotidyltransferase activity in MAB21L1 and its mutant form Arg51Leu purified protein. OAS1 protein purified in the same way is a positive control and when incubated with ATP and double-stranded RNA (dsRNA), significant pyrophosphate release is detected indicating nucleotidyl transferase activity. MAB21L1 and Arg51Leu showed no activity with either ATP, CTP, GTP, UTP used as substrate separately or as an equal mixture of NTPs using DNA or RNA as an activator. The error bars represent standard errors.B: Cellular fractionation: Western blot analysis of cytoplasmic (C) and nuclear(N) extracts from HEK293-Flp-In cells with Tetracyclin (TET) inducible expression of GFP-tagged wild-type and mutant MAB21L1(Arg51Leu and Arg51Gln).Wild type and mutant proteins were present in cytoplasm(C) as well as nuclear fraction(N) as detected by anti-GFP antibody. Representative Coomassie stain gel image is shown.C: Differential Gene Expression: Gene expression analysis by RNA Sequencing performed on GFP-tagged wild-type and mutant MAB21L1 (Arg51Leu and Arg51Gln) cells. Heatmap showing top 20 differentially expressed genes in the datasets (padj 1). The RNA sequencing data is available under the GSE166078 series at the NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). D: Effect of PAX6 overexpression on SPARC transcripts levels: SPARC transcripts levels were quantified using quantitative RT-PCR using cells expressing GFP tagged Wild type and mutant MAB21L1 with or without overexpressing PAX6. GAPDH transcripts levels were used as normalization control. The levels of SPARC transcripts were significantly reduced in GFP tagged Wild type MAB21L1 cells in presence of overexpressed PAX6. There was no significant difference in the mutant cells in presence or absence of overexpressed PAX6.</p

Supplemental clinical descriptions.

(DOCX)</p