Fig 1: ADAM33 contributes to the pathogenesis of thyroid cancer A The expression level of ADAM33 in the MDA-T32 and BCPAP cells was determined by real-time PCR. Cells were treated with doxycycline at a dose of 0.1 ug/ml for 2 days. GAPDH was used as the internal control in real-time PCR. N = 6, ***P < 0.001 by student’s t test. B The protein level of ADAM33 in the MDA-T32 and BCPAP cells was determined by Western blot. Cells were treated with doxycycline at a dose of 0.1 ug/ml for 2 days. GAPDH was used as the internal control. N = 3, ***P < 0.001 by student’s t-test. C CCK-8 assay determined the cell viability of MDA-T32 and BCPAP cells. Cells in A were treated with doxycycline at a dose of 0.1 ug/ml since they were seeded. Every two days, the culture medium was changed. N = 3, **P < 0.01 and ***P < 0.001 by two-way ANOVA. D The expression level of ADAM33 in the MDA-T32 and BCPAP cells was determined by real-time PCR. #1/2/3 indicates an independent shRNA target. GAPDH was used as an internal control in real-time PCR. Scr, scramble shRNA; N = 6, n.s, no significance and ***P < 0.001 by one-way ANOVA. E The protein level of ADAM33 in the MDA-T32 and BCPAP cells was determined by Western blot. #1/2/3 indicates an independent shRNA target. GAPDH was used as an internal control in Western blot. Scr, scramble shRNA; N = 3, ***P < 0.001 by one-way ANOVA. F CCK-8 assay determined the cell viability of MDA-T32 and BCPAP cells. Cells in C were used. Every two days, the culture medium was changed. N = 3, **P < 0.01 and ***P < 0.001 by two-way ANOVA. G-H. Colony formation ability of ADAM33 down- and over-expressed MDA-T32 and BCPAP cells. MDA-T32 and BCPAP cell lines in A and C (103 per well) were seeded in six-well plates. Every 3 days, the medium in each well was changed until the visible colonies formed. N = 6; n.s, no significance, ***P < 0.001 by one-way ANOVA
Fig 2: ADAM33 short isoform interfere the oncogenic function of full length ADAM33. A-B The expression level of ectopic ADAM33 B/ADAM33-n C in the MDA-T32 and BCPAP cells we collected was determined by real-time PCR. GAPDH was used as an internal control in real-time PCR. VEC, empty vector; N = 6, ***P < 0.001 by one-way ANOVA. C CCK-8 assay determined the cell viability of MDA-T32 and BCPAP cells. Cells in B and C were used. Every two days, the culture medium was changed. N = 3,***P < 0.001 by two-way ANOVA. D Colony formation ability of ADAM33 down- and over-expressed MDA-T32 and BCPAP cells. MDA-T32 and BCPAP cell lines in B and C (103 per well) were seeded in six-well plates. Every 3 days, the medium in each well was changed until the visible colonies formed. N = 6; ***P < 0.001 by one-way ANOVA. E. The expression of ADAM33-n when ADAM33 was overexpressed or knocked down. VEC, empty vector; N = 6, n.s, no significance by one-way ANOVA. F. The expression level of endogenous ADAM33 in the MDA-T32 and BCPAP cells we collected was determined by real-time PCR. Cells were stably over-expressed with ADAM33-n in a concentration gradient. GAPDH was used as an internal control in real-time PCR. VEC, empty vector; N = 6, n.s, no significance by one-way ANOVA
Fig 3: ADAM33 short isoform exhibits anti-oncogenic roles in thyroid cancer. A The expression level of ADAM33-n in the MDA-T32 and BCPAP cells was determined by real-time PCR. Cells were treated with doxycycline at a dose of 0.1 ug/ml for 2 days. GAPDH was used as an internal control in real-time PCR. N = 6, ***P < 0.001 by student’s t test. B CCK-8 assay determined the cell viability of MDA-T32 and BCPAP cells. Cells in A were treated with doxycycline at a dose of 0.1 ug/ml since they were seeded. Every two days, the culture medium was changed. N = 3, *P < 0.05 and ***P < 0.001 by two-way ANOVA. C The location of ADAM33-n specific shRNAs. CDS, coding sequence, 3’-UTR, 3’-untranslated region. D The expression level of ADAM33 in the MDA-T32 and BCPAP cells we collected was determined by real-time PCR. #1/2/3 indicates an independent shRNA target. GAPDH was used as an internal control in real-time PCR. Scr, scramble shRNA; N = 6, n.s, no significance and ***P < 0.001 by one-way ANOVA. E CCK-8 assay determined the cell viability of MDA-T32 and BCPAP cells. Cells in D were used. Every two days, the culture medium was changed. N = 3, *P < 0.05 and **P < 0.01 by two-way ANOVA. F-G. Colony formation ability of ADAM33 down- and over-expressed MDA-T32 and BCPAP cells. MDA-T32 and BCPAP cell lines in A and D (103 per well) were seeded in six-well plates. Every 3 days, the medium in each well was changed until the visible colonies formed. N = 6; n.s, no significance, **P < 0.01 and ***P < 0.001 by one-way ANOVA
Fig 4: ADAM33 interacts with ADAM33-n. A Crystal structure of ADAM33 catalytic domain (left) and the putative mechanism of ADAM33-n interfering full-length ADAM33 function. The carton of ADAM33 catalytic was generated based on the published structure in the PDB database (1R54). B Co-immunoprecipitation of HA-tagged ADAM33-n with ADAM33 in MDA-T32 and BCPAP cells
Fig 5: A novel isoform of ADAM33 is aberrantly expressed in thyroid cancer samples. A The expression level of ADAM33 in 53 different human tissues. The expression data are derived from the GTEx database. TPM, Transcripts Per Million mapped reads. B The usage of ADAM33 exons in 53 different human tissues. The expression data are derived from the GTEx database. C The expression pattern of ADAM33 isoforms in 53 different human tissues. The expression data are derived from the GTEx database
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