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

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

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

Sociodemographics, Risk Behaviors, and Sexual Network Characteristics of Community-Recruited Black MSM Stratified by Self-Reported HIV Serostatus and Age, Participant- and Network-Level Data (N = 1,349 Participants).

<p>Numbers may not add up to total due to missing data.</p><p>NA: Not applicable.</p><p>^a P-value <0.05 for all variables when compared by study city, except for self-reported HIV serostatus at enrollment. Site-specific differences may be reflective of different recruitment strategies used by the study sites, rather than of overall differences between cities.</p><p>^b P-value comparing self-reported HIV-positive men with self-reported HIV-negative men</p><p>^c SDUI: serodiscordant/serostatus unknown unprotected anal and/or vaginal intercourse</p><p>^d Age category difference is based on the following age categorical variables of the sexual partners in the social and sexual network inventory: ≤17 years, 18–20, 21–25, 25–30, 30–40, 40–50, 50–60, and ≥60</p><p>Sociodemographics, Risk Behaviors, and Sexual Network Characteristics of Community-Recruited Black MSM Stratified by Self-Reported HIV Serostatus and Age, Participant- and Network-Level Data (N = 1,349 Participants).</p

Sex Partner Characteristics, Condom Use, and HIV Serostatus Disclosure of Community-Recruited Black MSM, Partner-Level Data (N = 4,449 Partners).

<p>Numbers may not add up to column total due to missing data.</p><p>^a P-value <0.05 for all variables compared by study city. Site-specific differences may be reflective of different recruitment strategies used by the study sites, rather than of overall differences between cities.</p><p>Sex Partner Characteristics, Condom Use, and HIV Serostatus Disclosure of Community-Recruited Black MSM, Partner-Level Data (N = 4,449 Partners).</p

Participant and Sex Network Characteristics Associated with Having a Black Sex Partner, Having a Partner with at Least 2 Age Category Difference, and Having SDUI in the Last 6 Months among Community-Recruited Participants Stratified by Self-Reported HIV Serostatus of Participant, Multivariate GEE Models (N = 4,449 Partners).

<p>GEE: Generalized estimating equation, SDUI: serodiscordant/serostatus unknown unprotected anal and/or vaginal intercourse, AOR: Adjusted odds ratio, 95% CI: 95% Confidence Interval, Ref: Reference, NA: Not applicable as there were no observations detected.</p><p>Participant and Sex Network Characteristics Associated with Having a Black Sex Partner, Having a Partner with at Least 2 Age Category Difference, and Having SDUI in the Last 6 Months among Community-Recruited Participants Stratified by Self-Reported HIV Serostatus of Participant, Multivariate GEE Models (N = 4,449 Partners).</p