Figure 1.
Pathway to show how Mcl-1, a member of the Bcl-2 family, influences apoptosis.
(A) The intrinsic (mitochondrial) cell death pathway is controlled by the Bcl-2 protein family, including BH3-only proteins, anti-apoptotic Bcl-2 proteins, Bax, Bak and Bid. (B) The Mcl-1 gene consists of three exons; the anti-apoptotic Mcl-1L and the pro-apoptotic Mcl-1S protein isoforms result from inclusion and skipping of exon 2 (open box), respectively.
Figure 2.
Expression of Mcl-1 and SR proteins in MCF-7 and JAR cells.
Top panel shows Mcl splice isoforms in commercially available JAR cell lysate, MCF-7 cells and JAR cells. Detection by western blotting of Mcl-1 splice variants, SRSF1-6 and the loading control GAPDH in 40 ug of total cell lysate from MCF-7 and JAR cells.
Table 1.
siRNA sequences.
Figure 3.
Knockdown of RNA binding proteins in MCF-7 cells and their effect on the mRNA levels of Mcl-1 splice isoforms.
MCF-7 cells were transfected with SRSF1-6, Tra2β, SREK1, GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were left untreated (UT). (A) Semi-quantitative RT-PCR showing both Mcl-1 spliced variants. (B) Semi-quantitative RT-PCR showing knockdown of RNA binding proteins 48 hours after transfection with siRNAs. (C) Semi-quantitative RT-PCR showing levels of the Mcl-1 splice isoforms (Mcl-1L and Mcl-1S) and the loading control GAPDH 72 hours after transfection with siRNAs. (D) Mcl-1L levels measured by real-time PCR on the same sample shown in (B). (E) Mcl-1S levels measured by real-time PCR on the same sample shown in (B). (F Mcl-1S levels in sample replicates measured by real-time PCR (mean (n = 3) ± SEM). ** P≤0.01; * P≤0.01.
Table 2.
Primer sequences.
Figure 4.
Knockdown of RNA binding proteins in JAR cells and their effect on the mRNA levels of Mcl-1 splice isoforms.
JAR cells were transfected with SRSF1-6, Tra2β, SREK1, GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were left untreated (UT). (A) Semi-quantitative RT-PCR showing knockdown of RNA binding proteins 48 hours after transfection with siRNAs. (B) Semi-quantitative RT-PCR showing levels of the Mcl-1 splice isoforms (Mcl-1L and Mcl-1S) and the loading control GAPDH 72 hours after transfection with siRNAs. (C) Mcl-1L levels measured by real-time PCR on the same sample shown in (B). (D) Mcl-1S levels measured by real-time PCR on the same sample shown in (B). (E) Mcl-1S levels in sample replicates measured by real-time PCR (mean (n = 3) ± SEM). * P≤0.01.
Figure 5.
Knockdown of RNA binding proteins and their effect on the protein levels of Mcl-1 splice isoforms.
(A) MCF-7 cells were transfected with SRSF1 (1 and 2), SRSF5 (1 and 2), GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were untreated (UT). SRSF1, 5, Mcl-1 and GAPDH protein levels were determined by immunoblotting 72 hours after transfection with the siRNAs, using 30µg of total cell lysate. Histograms show densitometric analysis of Mcl-1L and Mcl-1S protein expression. Data are shown as the mean ± SEM. ** indicates P≤0.01; * indicates P<0.05. (B) JAR cells were transfected with SRSF1 (1 and 2), GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were untreated (UT). SRSF1, Mcl-1 and GAPDH protein levels were determined by immunoblotting 72 hours after transfection with the siRNAs, using 30 µg of total cell lysate. (C) After transfection with SRSF1 (1) and NC siRNA MCF-7 cells were treated with 200 µM etoposide or DMSO control for 6 hours. Induction of apoptosis was assessed by measuring caspase3/7 activity.* indicates P<0.05.
Figure 6.
Effect of SRSF1 knockdown on expression, stability and translation of Mcl-1L in MCF-7 and JAR cells.
(A) MCF-7 and (B) JAR cells were transfected with SRSF1 (1 and 2) and negative control (NC) siRNA. Three days after transfection cells were treated with 35 ug/ml cycloheximide and protein samples were collected after 0, 0.5, 1, 1.5, 2, 3, 5 and 7 hours to assess protein stability. Mcl-1L protein levels were then measured by immunoblotting. (C) MCF-7 cells were transfected with SRSF1 (1 and 2), GAPDH (G) and negative control (NC) siRNA, or treated with vehicle (lipid) only (V), or were untreated (UT). 48 hours after transfection cells were treated with 20 nM rapamycin for a further 24 hours. Western blotting was then used to assess protein levels of SRSF1, Mcl-1L and GAPDH. (D) mir-29b levels measured by real-time PCR in JAR and MCF-7 cell lines (mean (n = 3) ± SEM), ** P≤0.01. Knockdown of SRSF1 in MCF-7 cells (E) and JAR cells (F) was assessed by semi-quantitative RT-PCR in SRSF1 (1) and negative control (NC) siRNA (upper panels). Levels of mir-29b were measured by real-time PCR in SRSF1 (1) and negative control (NC) siRNA treated MCF-7 cells from (E) and JAR cells from (F) along with GAPDH (G) siRNA, vehicle (lipid) only (V), and untreated (UT) cells (lower panels) (mean (n = 3) ± SEM).
Figure 7.
Effect of SRSF1 and SRSF5 knockdown on expression, stability and translation of Mcl-1L in MDA-MB-231 cells.
(A) Detection of Mcl-1 splice variants and GADPH proteins in MCF-7 and MDA-MB-231 using 40 ug of total cell lysate from both cell types. Histograms show densitometric analysis of Mcl-1L and Mcl-1S protein expression. Data are shown as the mean ± SEM. (B) Semi-quantitative RT-PCR showing knockdown of RNA binding proteins 48 hours after transfection with siRNAs. MDA-MB-231 cells were transfected with SRSF1, GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were left untreated (UT). Semi-quantitative RT-PCR showing levels of the Mcl-1 splice isoforms (Mcl-1L and Mcl-1S) and the loading control GAPDH 72 hours after transfection with siRNAs. Mcl-1S levels in sample replicates measured by real-time PCR (mean (n = 3) ± SEM). (C) MDA-MB-231 cells were transfected with SRSF1, SRSF5, GAPDH (G) and Negative control (NC) siRNAs, or treated with vehicle (lipid) only (V), or were untreated (UT). SRSF1, 5, Mcl-1 and GAPDH protein levels were determined by immunoblotting 72 hours after transfection with the siRNAs, using 30 µg of total cell lysate. (D) MDA-MB-231 cells were transfected with SRSF1, GAPDH (G) and negative control (NC) siRNA, or treated with vehicle (lipid) only (V), or were untreated (UT). 48 hours after transfection cells were treated with 20 nM rapamycin for a further 24 hours. Immunoblotting was then used to assess protein levels of SRSF1, Mcl-1L and GAPDH (mean (n = 3) ± SEM). *** indicates P≤0.001; ** P≤0.01.