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. 2020 Jan 20;48(5):2502–2517. doi: 10.1093/nar/gkz1237

Figure 2.

Figure 2.

Stress induces expression of the lncRNA LASTR in a JNK/c-JUN-dependent manner. (A) Graphical representation of the LASTR-1 promoter region along with the ENCODE CHIP-seq data from Hela-S3, HUVECs and K562 cells summarized in a black bar. The red bar shows a canonical c-JUN binding site. (B) Boxplot showing LASTR expression in TCGA BRCA patients stratified by levels of c-JUN phosphorylated at Ser73. Median was used to define high and low levels of phospho-c-JUN. P‐value was determined by a Mann–Whitney test. n = number of patients. (C) Immunoblot analysis of phosphorylated and total c-JUN in MCF10A cells treated with DMSO or SP600125 (20 μM, 18 h). (D) RT-qPCR analysis of LASTR expression in MCF10A cells treated with DMSO or SP600125 (20 μM) for the indicated time periods, n = 3. (E) Immunoblot analysis of phosphorylated and total c-JUN in MCF10A cells treated with DMSO or SP600125 (20 μM, 18 h) under normoxia or hypoxia (1% O2, 16 h). (F) RT-qPCR analysis of LASTR expression in normoxic or hypoxic (1% O2, 16 h) MCF10A cells treated for 18 h with DMSO or SP600125 (20 μM), n = 3. (G) Wild-type LASTR and mutant LASTR promoter activity in MCF10A cells treated with DMSO or SP600125 (20 μM, 18 h) under normoxia or hypoxia (1% O2, 16 h) as determined by a dual-luciferase reporter assay. P-value was determined by a one-way ANOVA test, n = 3. (H) Immunoblot analysis of phosphorylated and total c-JUN in MCF10A cells treated for 1 h with DMSO or doxorubicin (0.3 μM). (I) RT-qPCR analysis of LASTR expression in MCF10A cells treated for 24 h with DMSO or doxorubicin (0.3 μM). n = 3. (J) RT-qPCR analysis of LASTR expression in MCF10A cells treated for 24 h with doxorubicin (0.3 μM), or the combination of doxorubicin (0.3 μM) and SP600125 (20 μM), n = 3 (K) qPCR analysis of LASTR promoter recovered in ChIP assays conducted with the indicated antibodies. Chromatin precipitation was performed using MCF10A cells under normoxia or hypoxia (1% O2, 16 h), n = 3. (D, F, I, J, K) Data are presented as mean ± s.e.m.; P-values were determined by two-sided t-tests.