Figure 4.
Figure 4. Role of SENP1 in CMS polycythemia in the Andean population. (A) The SENP1 region in Andean highlanders. Four known transcripts of human SENP1 with accession nos. (NCBI RefSeq) are shown. Note that SENP1 is transcribed from the negative strand (i.e., right to left). Overlaid above in blue are the genomic positions of 66 SNPs deemed differential by Zhou et al. (2013). Three of our differential SNPs (marked with black arrows) overlap with different regulatory regions such as promoters and enhancers, as described in the Critical role of SENP1 in polycythemia section of Results. These SNPs show a strong signal of frequency differentiation between the non-CMS and CMS highlanders, indicative of strong positive selection in the region. (B) Western blot analysis of SENP1 protein expression under hypoxia (5% O2) and normoxia (21% O2) for CMS and non-CMS groups. The representative blot is shown is from one experiment. The relative levels were computed for n = 5 for CMS and n = 4 for non-CMS under hypoxia and normoxia. Data represent at least two to three measurements. The experiment was repeated at least twice for each subject. (C) iPSCs were infected with lentivirus and selected by puromycin. shSENP1#1, shSENP1#2, and shSENP1#3 represent the three clones selected. Each clone showed significant down-regulation of Senp1 expression by quantitative PCR, compared with uninfected iPSCs, as well as an iPSC line infected by control vector. Data represent two measurements in duplicate. (D) Western blot analysis of the loss of SENP1 expression in the shRNA clones. Lanes 1 and 2 show significant reduction in SENP1 levels in shRNA clones 1 and 2 as compared with uninfected and scrambled controls. Each bar represents the mean ± SEM of at least two to three measurements. The experiment was repeated at least two times for each subject. (E) Loss of vigorous erythropoietic response of CMS cells to hypoxia. The first bar represents the response of CMS cells to hypoxia. CMS-Senp1-shRNA represents the CMS cells that were infected by shRNA of Senp1 using lentivirus infection. Four different clones were tested for each line. Data represent three measurements in triplicates. The experiment was repeated three times. An unpaired Student’s t test was used. (F) Overexpression of SENP1 in non-CMS iPSCs. The blot shows a representative image for one experiment. We observed a twofold increase in expression in non-CMS in the cDNA overexpression cell line. Data represent three measurements in triplicates. The experiment was repeated twice. An unpaired Student’s t test was used. (G) SENP1 overexpression (OE) leads to marked increase in RBCs in non-CMS erythroid cell differentiation. The scrambled control overexpression did not change the phenotype of CMS. Each bar represents the mean for each clone measured in triplicates. The experiment was repeated three times. Error bars represent the mean ± SEM. *, P < 0.05; ***, P < 0.001.

Role of SENP1 in CMS polycythemia in the Andean population. (A) The SENP1 region in Andean highlanders. Four known transcripts of human SENP1 with accession nos. (NCBI RefSeq) are shown. Note that SENP1 is transcribed from the negative strand (i.e., right to left). Overlaid above in blue are the genomic positions of 66 SNPs deemed differential by Zhou et al. (2013). Three of our differential SNPs (marked with black arrows) overlap with different regulatory regions such as promoters and enhancers, as described in the Critical role of SENP1 in polycythemia section of Results. These SNPs show a strong signal of frequency differentiation between the non-CMS and CMS highlanders, indicative of strong positive selection in the region. (B) Western blot analysis of SENP1 protein expression under hypoxia (5% O2) and normoxia (21% O2) for CMS and non-CMS groups. The representative blot is shown is from one experiment. The relative levels were computed for n = 5 for CMS and n = 4 for non-CMS under hypoxia and normoxia. Data represent at least two to three measurements. The experiment was repeated at least twice for each subject. (C) iPSCs were infected with lentivirus and selected by puromycin. shSENP1#1, shSENP1#2, and shSENP1#3 represent the three clones selected. Each clone showed significant down-regulation of Senp1 expression by quantitative PCR, compared with uninfected iPSCs, as well as an iPSC line infected by control vector. Data represent two measurements in duplicate. (D) Western blot analysis of the loss of SENP1 expression in the shRNA clones. Lanes 1 and 2 show significant reduction in SENP1 levels in shRNA clones 1 and 2 as compared with uninfected and scrambled controls. Each bar represents the mean ± SEM of at least two to three measurements. The experiment was repeated at least two times for each subject. (E) Loss of vigorous erythropoietic response of CMS cells to hypoxia. The first bar represents the response of CMS cells to hypoxia. CMS-Senp1-shRNA represents the CMS cells that were infected by shRNA of Senp1 using lentivirus infection. Four different clones were tested for each line. Data represent three measurements in triplicates. The experiment was repeated three times. An unpaired Student’s t test was used. (F) Overexpression of SENP1 in non-CMS iPSCs. The blot shows a representative image for one experiment. We observed a twofold increase in expression in non-CMS in the cDNA overexpression cell line. Data represent three measurements in triplicates. The experiment was repeated twice. An unpaired Student’s t test was used. (G) SENP1 overexpression (OE) leads to marked increase in RBCs in non-CMS erythroid cell differentiation. The scrambled control overexpression did not change the phenotype of CMS. Each bar represents the mean for each clone measured in triplicates. The experiment was repeated three times. Error bars represent the mean ± SEM. *, P < 0.05; ***, P < 0.001.

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