Additional file 2 of Long non-coding RNA NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of esophageal squamous cell carcinoma by promoting nuclear accumulation of β-catenin

Additional file 2: Figure S1. The effect of CDDP on cell viability of ESCC cells. a The effect of CDDP on cell viability of KYSE30/CDDP-R and KYSE30 cells. b The effect of CDDP on cell viability of TE1/CDDP-R and TE1 cells. Figure S2. Representative images of RNA FISH of NORAD in TE1/CDDP-R and TE1 cells (× 1000), which show that NORAD is predominantly located in the cytoplasm. Nuclei are stained with DAPI. Figure S3. The effect of sh-NORAD and overexpression vector on the expression of NORAD and proliferation ability of ESCC cells. a The effect of sh-NORAD on NORAD expression in KYSE30/CDDP-R and TE1/CDDP-R cells. Error bars denote SD of triplicates. ***P < 0.001. b The effect of overexpression of NORAD on NORAD expression in KYSE30 and TE1 cells. Error bars denote SD of triplicates. ***P < 0.001. c The effect of sh-NORAD on proliferation ability of KYSE30/CDDP-R and TE1/CDDP-R cells. d The effect of overexpression of NORAD on proliferation ability of KYSE30 and TE1 cells. The results are presented as the mean ± SD. ***P < 0.001. Figure S4. NORAD contributes to CDDP resistance of TE1 cells. a NORAD knockdown increases the sensitivity of TE1/CDDP-R cells to CDDP, detected by CCK-8. b Overexpression of NORAD decreases the sensitivity of TE1/CDDP-R cells to CDDP, detected by CCK-8. c NORAD knockdown decreases the colony formation ability of TE1/CDDP-R cells in the presence of CDDP. d Overexpression of NORAD increases the colony formation ability of TE1 cells in the presence of CDDP. e NORAD knockdown increases the CDDP-induced apoptosis rate of TE1/CDDP-R cells, and overexpression of NORAD decreases the CDDP-induced apoptosis rate of TE1 cells, detected by FCM. f NORAD knockdown facilitates CDDP to induce cell cycle arrest in TE1/CDDP-R cells. g Overexpression of NORAD suppresses CDDP-induced cell cycle arrest in TE1 cells. h NORAD knockdown increases the CDDP-induced γH2AX and cleaved caspase-3 in TE1/CDDP-R cells. i Overexpression of NORAD decreases the CDDP-induced γH2AX and cleaved caspase-3 of TE1 cells. In all cases, error bars denote SD of triplicates. ***P < 0.001. Figure S5. MTDH is a direct target of miR-224-3p in TE1 cells. a Representative images of RNA FISH of NORAD and miR-224-3p in TE1/CDDP-R cells, which show that NORAD and miR-224-3p are co-located in the cytoplasm. Nuclei are stained with DAPI. b The nuclear and cytoplasmic expression of NORAD and miR-224-3p in TE1/CDDP-R cells. c The structure of the luciferase reporter plasmid. The sequences of MTDH-3′-UTR-WT or MTDH-3′-UTR-Mut were subcloned into the cloning region. d Luciferase reporter assay showing the luciferase activity of MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 and MTDH-3′-UTR-Mut#4 in TE1/CDDP-R cells co-transfected with miR-224-3p mimic. e The effects of miR-224-3p mimic on the expression of MTDH in TE1/CDDP-R cells transfected with MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 or MTDH-3′-UTR-Mut#4. f NORAD knockdown downregulates MTDH expression in TE1/CDDP-R cells and miR-224-3p mimic rescues this downregulation. g Overexpression of NORAD upregulates MTDH expression in TE1 cells and miR-224-3p inhibitor rescues this downregulation. b-e Error bars denote SD of triplicates. ***P < 0.001. Figure S6. a Predicted binding sites on NORAD to sponge miR-224-3p and matched established mutant sequences for luciferase reporter assay. b The structure of the luciferase reporter plasmid. The sequences of NORAD-WT or NORAD-Mut were subcloned into the cloning region. Figure S7. The NORAD/miR-224-3p/MTDH axis promotes CDDP resistance in TE1 cells by promoting nuclear accumulation of β-catenin. a The effects of NORAD/miR-224-3p/MTDH axis on the colony formation ability of TE1/CDDP-R and TE1 cells in the presence of CDDP. b The effects of NORAD/miR-224-3p/MTDH axis on CDDP induced apoptosis of TE1/CDDP-R and TE1 cells. c NORAD knockdown facilitates CDDP to induce cell cycle arrest in TE1/CDDP-R cells while miR-224-3p inhibitor rescued the arrest. d Overexpression of NORAD suppresses CDDP-induced cell cycle arrest in TE1 cells while miR-224-3p mimic neutralizes the suppression. e The effects of NORAD/miR-224-3p/MTDH axis on the expression and phosphorylation of MAPK, Akt, NF-κB and β-catenin in TE1/CDDP-R cells. f NORAD knockdown decreases the nuclear expression of MTDH in TE1/CDDP-R cells, detected by western blotting. g NORAD knockdown decreases the nuclear expression of MTDH in TE1/CDDP-R cells, detected by immunofluorescence. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001. Figure S8. The NORAD/miR-224-3p/MTDH axis promotes progression of TE1 cells. a The effects of the NORAD/miR-224-3p/MTDH axis on migratory ability of TE1/CDDP-R and TE1 cells. b The effects of the NORAD/miR-224-3p/MTDH axis on invasive ability of TE1/CDDP-R and TE1 cells. c The effects of the NORAD/miR-224-3p/MTDH axis on the expression of E-cadherin, N-cadherin and MMP9 in TE1/CDDP-R and TE1 cells. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001.