Fig. 5: Phosphorylation of ACSL4 at T679 site is critical for ACSL4 oncogenic function through Src myristoylation via Src/ERK pathway. | Nature Communications

Fig. 5: Phosphorylation of ACSL4 at T679 site is critical for ACSL4 oncogenic function through Src myristoylation via Src/ERK pathway.

From: p38 mediated ACSL4 phosphorylation drives stress-induced esophageal squamous cell carcinoma growth through Src myristoylation

Fig. 5

a The production of C14:0 CoA in KYSE150 and KYSE70 cells transfected with ACSL4 knock down or sh-Mock as control group (n = 3 biological replicates, mean ± SD). P values were determined by One-way ANOVA. b KYSE150 and KYSE70 cells transfected with shRNA-Mock, shRNA-ACSL4#1, or shRNA-ACSL4#5 by lentiviral infection were cultured with 50 μM myristic acid-alkynyl (Alk-myr) overnight. The protein was extracted, and Src was enriched using anti-Src antibody. Then Src myristoylation in samples was analyzed by Click Chemistry, and detected by streptavidin-HRP (SA-HRP) (c) The production of C14:0 CoA in KYSE450 cells transfected with vector, ACSL4-flag, ACSL4-T679A-flag, and ACSL4-T679D-flag (n = 3 biological replicates, mean ± SD). P values were determined by One-way ANOVA. d Src myristoylation in KYSE450 cells transfected with vector, ACSL4-flag, ACSL4-T679A-flag, and ACSL4-T679D-flag were analyzed by Click Chemistry, and detected by streptavidin-HRP. (e, left) Src/ERK pathway was detected after ACSL4 knock down. (e, right) Src/ERK pathway was detected after KYSE450 cells transfected with vector, ACSL4-flag, ACSL4-T679A-flag, and ACSL4-T679D-flag. f KYSE150 and KYSE70 cells were treated with 4NQO or heat stress to examine the change of p-p38 (T180/Y182), p-ACSL4 T679, and p-ERK1/2 (T202/Y204). g The samples were harvested and stained with IHC analysis of p-ACSL4 (T679) at different time points in 4NQO-induced ESCC progression (n = 3 biological replicates, mean ± SD). Statistical significance was conducted by two-sided unpaired Student’s t-test. h KYSE150 and KYSE70 cells were subjected to SB202190 followed by treatment with heat stress and examined the change of p-p38 (T180/Y182), p-ACSL4 (T679), and p-ERK1/2 (T202/Y204) by Western blot. i Western blots on KYSE150 and KYSE70 cells with ACSL4 knockdown treated with LPS (p38 agonist) to observe the levels of p-p38 (T180/Y182), p-ACSL4 (T679), and p-ERK1/2 (T202/Y204). j p-p38, p-ACSL4 T679 and p-ERK1/2 (T202/Y204) was determined by Western blot in ACSL4 knockdown cells rescued with different ACSL4 T679 mutations. Representative immunoblots shown in figures were repeated three times independently with similar results. Source data are provided as a Source Data file.

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