PMC

a The expression of YTHDF1 in siYTHDF1 H1299 cells and control cells was determined by qRT-PCR assays. The results showed that the expression of YTHDF1 was downregulated in siYTHDF1 cells compared with that of the control cells (*p < 0.05, ***p < 0.001). b The expression of lncRNA THOR in siYTHDF1 H1299 cells and control cells was determined by qRT-PCR assays. The results showed that the expression of THOR was downregulated in siYTHDF1 cells compared with that of the control cells (*p < 0.05, ns denotes not significant). c The expression of YTHDF2 in siYTHDF2 H1299 cells and control cells was determined by qRT-PCR. The results showed that the expression of YTHDF2 was upregulated in siYTHDF2 cells compared with that of control cells (***p < 0.001). d The expression of lncRNA THOR in siYTHDF2 H1299 cells and control cells was determined by qRT-PCR. The results showed that the expression of lncRNA THOR was upregulated in siYTHDF2 cells compared with that of control cells (***p < 0.001). e The interaction between lncRNA THOR and YTHDF1 was determined by RIP-qPCR assay. Western blotting assays showed that the antibody was specific, and the interaction between lncRNA THOR and YTHDF1 was confirmed by qRT-PCR (*p < 0.05, **p < 0.01). f The interaction between lncRNA THOR and YTHDF2 was determined by RIP-qPCR assay. Western blotting assays showed that the antibody was specific, and the interaction between lncRNA THOR and YTHDF2 was confirmed by qRT-PCR (*p < 0.05, **p < 0.01).

A, Expression of THOR in TCGA tumors and normal tissue samples from GTEX and TCGA. B, Expression of THOR in the CCLE cell line panel. C, Expression of THOR in the TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) samples represented alongside each tissue’s matched normal samples. D, qRT-PCR validation in an independent tissue in lung adenocarcinoma (benign, n=13; cancer, n=180) and melanoma tissues (benign, n = 2; cancer, n=24). E, Expression levels of THOR in two melanoma (SKCM) and two non-small cell lung cancer (NSCLC) cell lines. Data show mean ± S.D. F, Cell proliferation assays for NCI-H1299 cells treated with 2 independent THOR siRNAs. G, Cell proliferation assays for NCI-H1299 cells treated with 2 independent THOR ASOs. H, Anchorage-independent growth of H1299 cells transfected with non-targeting siRNA (si-NT) or two THOR siRNAs (siTHOR-A, siTHOR-B). Left, quantification of number of colonies. Right, representative image of surviving colonies and individual colony. I, Cell proliferation assay for NCI-H1299 cells with CRISPR-Cas9 mediated THOR knockout vs control in the context of LacZ and THOR overexpression. J, THOR knockout NCI-H1299 cell line xenografts (N=10) demonstrate decreased tumor growth relative to control samples (N=10). Tumor volumes at each time point by caliper measurement are shown. K, Cell proliferation assay in NCI-H1437 cells stably transfected with THOR overexpression or LacZ control lentivirus. Data show mean ± S.E. from one of the two independent experiments. L, Anchorage-independent growth of LacZ or THOR overexpressing H1437 cells. M, THOR overexpressing NCI-H1437 cell line xenografts (N=10) demonstrate increased tumor growth relative to control LacZ samples (N=10). Tumor volumes at each time point by caliper measurement are shown. Asterisk (*) indicates P ≤ 0.001 by a two-tailed Student’s t-test. Data show mean ± S.E.M. from one of the two independent experiments. For all panels, asterisk (*) indicates P ≤ 0.01, (**) indicates P ≤ 0.001, (***) indicates P ≤ 0.0001 by a two-tailed Student’s t-test.

A, Table reporting the protein binding partners for THOR in four different experimental conditions of RNA pull-down analysis: zebrafish THOR added to human H1299 cell lysate (green), human THOR added to human H1299 cell lysate (blue), zebrafish THOR added to zebrafish embryo lysate (yellow), and human THOR added to zebrafish embryo lysate (red). All proteins bound in any condition are displayed in the table, and each dot represents binding in the respective condition. B, Immunoprecipitation western blotting analysis (IP-WB) for various components of the IGFBP complex and HuR as a negative control. C, qRT-PCR following RIP of IGF2BP1, IGF2BP2, IGF2BP3, STAU1, YBX1, HUR, and IgG in H1299 cells. Data show mean ± S.D. from one of the two independent experiments. D, In vitro RNA-protein binding assay. In vitro transcribed THOR added to purified myc-tagged proteins. THOR qRT-PCR was then performed following anti-myc pull-down. E, Schematic representation of human THOR, antisense-THOR (AS), and various deletion constructs generated to interrogate IGF2BP1 binding (left). Fragment sizes confirmed by PCR (right, top), and binding of each fragment to IGF2BP1 determined via pulldown of BRU-labelled RNA fragments (right, bottom) in H1299 cells. F, Schematic representation of zebrafish THOR constructs generated to study IGF2BP1 binding. Fragment sizes confirmed by PCR (right, top), and binding of each fragment to zebrafish igf2bp1 determined via pulldown of BRU-labelled RNA fragments (right, bottom) in 16 hpf embryos. Asterisk (*) indicates P ≤ 0.01 by a two-tailed Student’s t-test. Data show mean ± S.D. from one of the two independent experiments.

A, Estimation of THOR mRNA expression by qRT-PCR in human adult normal tissue panel. B, H&E stain of human testis at high magnification (400×) (right), and RNA-ISH of THOR in human testis (left). Various cells of the testis are labelled as follows: (1) spermatogonia, (2) spermatocytes, (3) spermatids, (4) mature spermatozoa, and (5) scattered Sertoli cells with a single central prominent nucleolus. THOR expression is observed in the spermatid and spermatocyte. C, Measurement of mouse THOR expression by qRT-PCR on an adult murine tissue panel (left) and embryos (right). D, Quantification of zebrafish THOR expression by qRT-PCR on a piscine tissue panel (left) and embryos (right).