The testis of SMA mice expresses high levels of <i>SMN2</i> full-length mRNA and protein.

<p>Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both <i>SMN2</i> full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of <i>SMN2</i> FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.</p

The levels of Tra2-β1 and ASF/SF2 decrease during testis cell primary culture, which is correlated with <i>SMN2</i> exon 7 splicing.

<p>(A) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect Tra2-β1, ASF/SF2, hnRNP A1, SRp30c, and hnRNP Q. A representative result of two independent experiments was shown. The result showed that the levels of Tra2-β1, ASF/SF2, and hnRNP A1 decreased dramatically from 2-hour to 96-hour cultures. (B) Total RNA was isolated from primary testis cells cultured for different time periods and subjected to qRT-PCR to detect <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA expression levels. The result showed that the mRNA levels of <i>Tra2-β1</i> and <i>ASF/SF2</i> decreased after longer cultures, which was consistent with the protein expression. Error bars represent standard deviation. (*) <i>P</i> < 0.01, compared with 2-hour cultured cells.</p

The testis expresses the highest level of Tra2-β1 among the tissues examined.

<p>Various tissues of 18–20-week-old type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to qRT-PCR to detect <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA levels. The result showed that among the tissues examined, the testis expressed the highest level of <i>Tra2-β1</i> mRNA, but not the highest level of <i>ASF/SF2</i> mRNA. (B) Proteins were extracted and subjected to Western blotting to detect Tra2-β1 and ASF/SF2 proteins. The result also showed that among the tissues examined, the testis expressed the highest level of Tra2-β1 protein, but not the highest level of ASF/SF2 protein.</p

Overexpression of Tra2-β1, but not ASF/SF2, increases <i>SMN2</i> exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.

<p>Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with <i>SMN2</i> minigene plasmid and <i>Tra2-β1</i> overexpression plasmid, <i>ASF/SF2</i> overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify <i>SMN2</i> minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased <i>SMN2</i> exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the vector control.</p

The levels of <i>SMN2</i> full-length mRNA and protein decrease during testis cell primary culture.

<p>(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify <i>SMN2</i> FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of <i>SMN2</i> FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that SMN protein level was high in 2-hour cultured cells and decreased dramatically in 96-hour cultured cells, consistent with the result of <i>SMN2</i> exon 7 splicing. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with 2-hour cultured cells.</p

Knockdown of Tra2-β1 decreases <i>SMN2</i> exon 7 inclusion in primary testis cells of SMA mice.

<p>Primary testis cells of SMA mice were cultured for 72 hours and then transfected with Tra2-β1 siRNA or negative control (NC) siRNA for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify <i>SMN2</i> FL and Δ7 mRNAs. The results show that knockdown of Tra2-β1 promoted <i>SMN2</i> exon 7 exclusion in primary testis cells of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the siNC control.</p

supplementary from Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice

supplementary material, figure, and tabl

Additional file 2: Table S1. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Dysregulated miRNAs in TNBC patients. (DOCX 13 kb

Additional file 3: Table S2. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Univariate and multivariate analysis of clinical features and four oncomiRs associated with overall survival. (DOCX 12 kb

Additional file 1: Figure S1. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Association between identified miRNAs and overall survival in TNBC and non-TNBC patients. A Identification of dysregulated miRNAs in TNBC compared with non-TNBC using Student t test. B Elevated expression of miR-301b, miR-181a-3p, miR-105, and miR-93 were individually associated with poor overall survival in 1095 non-triple negative breast cancer patients by Kaplan-Meier analysis. The correlation between indicated miRNAs and overall survival in (C) 204 TNBC patients and (D) 1095 non-TNBC patients was analyzed by Kaplan-Meier analysis. E Indicated miRNAs expression levels were examined in the independent cohort (GSE40267, N = 173), which contained 94 TNBC, 79 non-TNBC. Figure S2 miR-105 and miR-93-3p promote cellular migration but not proliferation. The effect of ectopic overexpression or silencing of indicated miRNAs on cell proliferation as determined by (A) colony-forming assay and (B) MTT assay. C The effect of ectopic overexpression or silencing of indicated miRNAs on cell migration ability as measured by the Boyden chamber transwell migration assay. D The miRNA-overexpressing HCC70 cells and miRNA silenced-BT-549 cells were seeded into matrigel-coated transwells to evaluate cell invasion in vitro. Figure S3 miR-105 and miR-93-3p confer cisplatin resistance. Cisplatin was administered with the indicated dose to cells with (A) ectopic overexpression or (B) silencing of indicated miRNAs prior to determining cell viability by the MTT assay. C Co-transfection with miR-105 and miR-93-3p antagomiRs in HCC1937 followed by measurement of the cisplatin response by MTT assay. D miR-105/93-3p co-silenced-BT-549 cells were seeded at 10,000 cells/ml into an ultra-low attachment plate for 10 days to evaluate mammosphere formation, as an indicator of stemness. Relative efficiency of mammosphere formation was measured in control and miR-105/93-3p-knockdown BT-549 cells. Figure S4 miR-105 and miR-93-3p activate Wnt/β-catenin signaling. A Bioinformatic analysis to identify potential miR-105 and miR-93-3p target genes. B The miR-105 and miR-93-3p associated mRNA profiling, paired with miRNA microarrays from the same patients were analyzed by IPA. C Ectopic overexpression of miR-105 or miR-93-3p and subsequent determination of β-catenin activity by the TOP/FOP reporter assay. D Immunoblotting was performed to measure levels of the indicated proteins in miR-105 or miR-93-3p manipulated TNBC cells. Figure S5 miR-93-3p/105 target to SFRP1 and decrease SFRP1 expression level. A Bioinformatic prediction of target genes for miR-105/93-3p involved in Wnt/b-catenin signaling. B IPA was performed to identify potential upstream regulators of Wnt/β-catenin that are modulated in TNBC with high expression of miR-105 or miR-93-3p. C RNA hybrid was performed to examine the binding energy between miR-105 or miR-93-3p with 3′-UTR of SFRP1. D Ectopic overexpression or silencing of indicated miRNAs followed by determination of SFRP1 protein levels by immunoblotting. Figure S6 miR-93-3p/105 can serve as biomarker for early TNBC patients and inversely correlated with SFRP1 in TNBC but not in non-TNBC patients. A The SFRP1 levels were determined in 12 non-TNBC N-T paired tissues by immunoblotting. B The contingency plot showed the distribution of SFRP1 in the indicated circulating miRNA levels. C Overall survival of 249 TNBC patients and 93 TNBC patients with chemotherapy, those were obtained from Kaplan-Meier plotter website were stratified with SFRP1 by Kaplan-Meier analysis. D Combination of circulating miR-93-3p/105 to evaluate the predictive power for stage I/II (N = 46) or stage III/IV (N = 21) stage of TNBC by ROC curve. (PDF 29884 kb