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. 2023 Apr 25;10(17):2207257. doi: 10.1002/advs.202207257

Figure 3.

Figure 3

LncRNA DDIT4‐AS1 regulates stability of DDIT4 mRNA via recruiting AUF1. a) The location of lncRNA DDIT4‐AS1 in MDA‐MB‐231 cells was determined by FISH assay. Cy3 labeled probes are red; DAPI‐stained nuclei are blue; and U6 served as the positive control (scale bar: 10 µm). b) The expression level of DDIT4‐AS1 in the subcellular fractions of MDA‐MB‐231 and BT549 cells was detected by qRT‐PCR. U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. c) DDIT4 is a protein encoding gene adjacent to DDIT4‐AS1 locus. Location information was obtained from the UCSC Genome Browser. d) The correlation of DDIT4‐AS1 and DDIT4 mRNA was analyzed using the relative expression level from CLEE dataset. e) QRT‐PCR was used to detect he expression of DDIT4‐AS1 and DDIT4 mRNA in 63 paired breast tumor/adjacent tissues. β‐actin used as the internal control. f) QRT‐PCR was used to detect the effect of DDIT4‐AS1 knockdown on the expression of DDIT4 in breast cancer cells. β‐actin was the internal control. g) Western blot was used to detect the effect of DDIT4‐AS1 knockdown on the expression of indicated proteins in breast cancer cells. h) Western blot showing LC3‐II/LC3‐I and DDIT4 levels in MDA‐MB‐231 cells under treatment. i) 2.5 µg mL−1 ActD were used to block mRNA transcription, and qRT‐PCR was used to detect the effect of DDIT4‐AS1 knockdown on the degradation rate of DDIT4 mRNA in MDA‐MB‐231. 18s was the internal control. j) 2.5 µg mL−1 ActD were used to block mRNA transcription. qRT‐PCR was used to detect the effect of DDIT4‐AS1 overexpression on the degradation rate of DDIT4 mRNA in MDA‐MB‐231. 18s was the internal control. k) RNA pull down followed by western blotting was done with DDIT4‐AS1 probe to verify the direct interaction between RBPs and DDIT4‐AS1 in MDA‐MB‐231. l) In MDA‐MB‐231 cells, RIP was performed using anti‐AUF1 and control IgG antibodies, followed by qRT‐PCR to examine the enrichment of DDIT4‐AS1 and U6. U6 served as negative control, ***p < 0.001. m) The schematic of full‐length, truncated DDIT4‐AS1 (F1‐F4). n) The interaction between truncated DDIT4‐AS1 and DDIT4 mRNA, shown by RNA pull‐down and qRT‐PCR in MDA‐MB‐231. o,p) RNA pull‐down and western blotting showing the interaction of full‐length and truncated DDIT4‐AS1 o) or DDIT4 mRNA p) with AUF1 in MDA‐MB‐231 cell lysates. q,r) RIP was performed to detect endogenous AUF1 binding to DDIT4 mRNA in MDA‐MB‐231 cells with DDIT4‐AS1 overexpression q) or knockdown r). *p <0.05.