Figure 6. FAM120A promotes efficient splicing of lipogenic genes by associating with transcriptional and splicing machineries.

(A, B) QPCR analysis of intron retention in LAM cells transfected with siRNAs targeting U1-70K or control (A), and FAM120A, SREBP1/2, SREBP1/2+FAM120A or control (B). Cells were serum starved overnight. N = 3. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. p-value was calculated between siNTC and siFAM120A (B). N = 3.

(C) Co-IP analysis of HEK293E cells. Immunoprecipitation was performed using anti-FAM120A antibodies. Cell lysates were treated with RNases before IP. 1% of total cell lysate was loaded as an input.

(D) Co-IP analysis of HEK293E cells expressing FAM120A-V5 and T7-SRSF1 (WT or mutants). 1% of total cell lysate was loaded as an input. Asterisk indicates a non-specific band.

(E) Co-IP analysis of HEK293E cells expressing Flag-FAM120A WT or ∆RBD. 1% of total cell lysate was loaded as an input. Asterisk indicates a non-specific band.

(F-H) Co-IP analysis of LAM (F) and MCF7 (G) cells serum starved overnight (to induce SREBP cleavage) and treated with SREBP cleavage inhibitor (25-HC, 10 µM), or SRPK inhibitor (SRPKIN-1, 5 µM) for 4 hr (F, G); HEK239E cells serum starved overnight followed by insulin stimulation (100 nM) (to induce SREBP cleavage) with or without 25-HC (10 µM) or SRPKIN-1 (5 µM) for 4 hr (H). Note that serum starvation decreases mTORC1 activity in HEK293E cells (H) but does not affect constitutively active mTORC1 activity in LAM and MCF7 cancer cells (F, G). Immunoprecipitation was performed using IgG or anti-FAM120A antibodies. 1% of total cell lysate was loaded as an input. SREBP1(P), precursor SREBP1; SREBP1(M), cleaved and matured SREBP1.

See also Supplemental Figure S5.