Figure 1.

Identification of lncRNA DDIT4‐AS1 as an important regulator of autophagy in TNBC cells. a) Heat maps showing the differentially expressed lncRNAs between control and EBSS‐treated MDA‐MB‐231 cells by limiting the difference ratio (|log2 (Fold change)|>1) and significance level (q_value<0.05). Three samples were used in each group. Colors correspond to the expression level indicated by the log2‐transformed scale bar below the matrix. Red and blue reflect Max and Min levels, respectively. b) Volcano plots showing the expression profiles of lncRNAs (up 460, down 117). c,d) MDA‐MB‐231 cells were treated with EBSS for 6 h and low glucose (LG) medium for 24 h, respectively, and the expression levels of lncRNAs were detected via qRT‐PCR analysis. β‐actin was the internal control. ***p < 0.001. e) Western blot showing the effects of lncRNAs silencing on LC3‐II/β‐actin levels in MDA‐MB‐231 cells treated with EBSS. f) Representative electron microscopy images and quantification of autophagic vacuoles in DDIT4‐AS1 silencing MDA‐MB‐231 cells treated with EBSS. Scale bar, 2 µm. ***p < 0.001. Arrows depict autophagosomes, and the nucleus is denoted by N. g) Confocal microscopy showing the effects of EBSS incubation on mCherry‐GFP‐LC3 dots distribution in MDA‐MB‐231 DDIT4‐AS1 knockdown cells 48 h after mCherry‐GFP‐LC3 plasmid transfection (scar bar: 10 µm), ***p < 0.001. h) qRT‐PCR was performed to measure the differential expression of DDIT4‐AS1 in multiple breast cancer cells and the mammary epithelial cell MCF10A. TNBC cells: MDA‐MB‐231, BT549, MDA‐MB‐453, MDA‐MB‐468, MDA‐MB‐436, HCC1806; ER positive cells: T47D, MCF‐7; HER2 positive cells: SKBR3; murine TNBC: 4T1. β‐actin was the internal control. i) MDA‐MB‐231 and BT549 cell lines with stable DDIT4‐AS1 silencing were constructed using two sequences, respectively. j) Western blot showing the effects of DDIT4‐AS1 knockdown on LC3‐II/β‐actin levels in MDA‐MB‐231 and BT549 cells treated with chloroquine (CQ).