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. 2017 Jul 3;9(9):1294–1313. doi: 10.15252/emmm.201607315

Figure 4. MG132 reduces progerin transcripts and splicing factor SRSF‐1 protein expression, while SRSF‐5 protein levels are increased.

Figure 4

  1. Upper panel, lamin A/C, progerin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) (MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (Baf. A1) (100 nM), caspase‐6 inhibitor (Casp‐6 inh.) (50 μM) or leptomycin B (Lept. B) (20 ng/ml). Lower panels, progerin expression levels in drug (s)‐treated cells relative to DMSO‐treated cells (well no. 1) were normalized to GAPDH values. (n = 5).
  2. Downregulation of progerin transcripts in response to MG132. Quantitative real‐time PCR analyses of progerin mRNA levels in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells (Control: “C”). (n = 3).
  3. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, lamin A, lamin C, actin, GAPDH and SRSF‐1 expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells “C”. Urea was used to lyse cells. (n = 4).
  4. Proteasome activities in HGPS fibroblasts treated with 5 μM MG132 relative to DMSO‐treated cells, (Control: untreated cells). (n = 3).
  5. MG132‐mediated SRSF‐5 accumulation and SRSF‐1 downregulation correlate with progerin clearance. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, SRSF‐5, GAPDH, SRSF‐1, LC3BI, and LC3BII expression in MG132‐treated HGPS cells up to 96 h and relative to DMSO‐treated cells (Control: “C”). The medium was replaced with new drug every 48 h. Urea was used to lyse cells. (n = 4).
  6. siRNA inactivation of SRSF‐1 reduces progerin levels in HGPS fibroblasts. HGPS fibroblasts were transfected with control siRNA or with siRNA specific for SRSF‐1 and 48 h later cells were treated for 24 h with DMSO control (−) or with (+) MG132 (5 μM). (n = 3).
  7. Left panels, caspase‐mediated downregulation of SRSF‐1, in addition to autophagy, contribute to progerin clearance. Western blotting evaluation of lamin A/C, progerin, actin and SRSF‐1 in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (−) or with (+) the indicated drug (s) [MG132 (5 μM), chloroquine (50 μM), bafilomycin A1 (100 nM) or pan‐caspase inhibitor (50 μM)]. Rihgt panels, progerin and SRSF‐1 expression levels relative to DMSO‐treated cells (well no. 1) were normalized to actin values using ImageJ software. (n = 6).
Data information: Results are expressed as mean ± SEM, Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001, experimental vs. control; the exact P‐values are indicated in Appendix Table S1.Source data are available online for this figure.