Figure 1

LPA₂ upregulation and LPA₃ downregulation correlate with attenuated cell proliferation and increased senescence in Progerin HEK293 cells. (a) Design of vectors expressing Lamin A and Progerin. Puromycin was applied as the selection marker to establish Progerin HEK293 stable cells. (b) PCR with primer targeting outside of the cryptic splicing site shows alternate 150‐nucleotide splicing of Lamin A mRNA in Progerin HEK293 cells. (c) Western blot with anti‐Lamin A/C (ab108595) and anti‐Progerin (sc‐81611) antibody shows expression of Progerin in Progerin HEK293 cells (d) Western blot with LPA receptor antibodies shows upregulation of LPA₂ and downregulation of LPA₃ in Progerin HEK293 cells. Actin was used as a loading control. (e) Cell number measurements show that, in both Lamin A and Progerin HEK293 cells, activating LPA₂ with 5 μM of GRI decreased the cell proliferation rate, but activating LPA₃ with 100 nM of OMPT increased the cell proliferation rate. (f) Representative images of senescence‐associated β‐gal staining assay and its quantified results. Treating Lamin A and Progerin HEK293 cells with 5 μM of GRI for 4 days increased the percentage of β‐gal‐positive cells in both cell lines. Treating Progerin HEK293 cells with 100 nM OMPT for 4 days reduced the percentage of β‐gal‐positive cells. (g) Knockdown of LPA₂ by siRNA (siLPA₂) reduced β‐gal‐positive Progerin HEK293 cells. Knockdown of LPA₃ by siRNA (siLPA₃) increased β‐gal‐positive cells. (h) Overexpression of LPA₃ decreased β‐gal‐positive cells. (i) Real‐time qPCR showed that treatment of 100 nM OMPT for 24 hr reduced mRNA level of p16 in Progerin HEK293 cells. (j) 100 nM OMPT for 24 hr reduced mRNA level of p21. (k) 100 nM OMPT for 24 hr reduced mRNA level of IL6. GAPDH was used as internal control. ANOVA and Student's t test; *p < .05, **p < .01, ***p < .001