Fig 2: HNRNPL-mediated circular RNA formation regulates ITGβ3.A. IMCR-seq results showing the differential expression of different circular RNAs in the MDA-MB-468-RR and in the shHNRNPL-1 cells. The blue dots highlight the top 5 differentially expressed circular RNAs whereas the other labels represent other circular RNAs that can function as ceRNAs B. A graph showing the let-7 binding score for each circular RNA in comparison to its abundance (based on ranking of circular RNAs from CPM values) in MDA-MB-468-RR cells. C. mRNA expression of the top ceRNAs identified by Circr in the MDA-MB-468-RR and HNRNPL-knockdown cells (n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). D. mRNA expression of the top ceRNAs in MDA-MB-468 transfected with empty vector and HNRNPL (n=3, mean ± SD, unpaired t-test two-tailed). E. mRNA expression of ITGβ3 after siRNA treatment of the ceRNAs, with a representative image of the surface expression of integrin β3 and quantification based on flow cytometry (n=3, mean ± SD, unpaired t-test two-tailed). F. Representative flow cytometry data showing the surface expression of integrin β3 in the HNRNPL knockdown cells transducing with an empty plasmid or circRAB12. The graphs quantify the fold change in integrin β3 surface expression in the HNRNPL knockdown cells after transducing them with an empty plasmid or circRAB12. n=3, mean ± SD, unpaired t-test two-tailed.

Fig 3: HNRNPL stabilizes ITGβ3 mRNA and regulates integrin β3 expression.A. The gene sets from ‘REACTOME’ that were upregulated in the 4T1-RR and MDA-MB-468-RR cells with the red signifying gene sets associated with RNA metabolism. The mRNA expression of HNRNPL based on RT-qPCR in the B. MDA-MB-468 cell lines (n=3, mean ± SD, unpaired t-test two-tailed) and the C. 4T1 cell lines (n=3, mean ± SD, unpaired t-test two-tailed). D. The protein expression of HNRNPL in MDA-MB-468-RR cells and the HNRNPL-knockdown cells (shHL-1 and shHL-2) and the transcript stability assay of ITGβ3 after 0,1,2, and 4 hours of actinomycin D treatment (n=3, mean ± SD, two-way ANOVA with Tukey’s multiple comparison tests). E. The protein expression of HNRNPL in 4T1-RR cells and the HNRNPL-knockdown cells (shHL-1 and shHL-2) and the transcript stability assay of ITGβ3 after 0,1,2, and 4 hours of actinomycin D treatment (n=3, mean ± SD, two-way ANOVA with Tukey’s multiple comparison tests). F. The surface expression of ITGβ3 in the MDA-MB-468-RR cells and the HNRNPL-knockdown cells (n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). G. The surface expression of ITGβ3 in the 4T1-RR cells and the HNRNPL-knockdown cells (n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). H. The western blot expression of FLAG in the MDA-MB-468 cells transfected with empty vector and FLAG-tagged HNRNPL. The surface expression of ITGβ3 in the MDA-MB-468 empty vector and HNRNPL overexpression cells (n=3, mean ± SD, unpaired t-test two-tailed).

Fig 4: NRF2 regulates HNRNPL transcription.A. A Venn diagram showing the transcription factor genesets that were upregulated based on GSEA of the RNA-seq comparing the radioresistant cell lines to the parental cell lines along with the transcription factors that were identified by ENCODE transcription factor ChIP-seq data to regulate HNRNPL. B. A correlation between NRF2 activity score compared to HNRNPL expression in breast cancer patient samples collected by TCGA (n=1108, Spearman correlation). C. Immunofluorescent images of the MDA-MB-468-RR and MDA-MB-468 cells with blue being the DAPI dye and green representing NRF2. Scale bar is 10um. A ratio comparing NRF2 localization in the nucleus vs the cytoplasm for the MDA-MB-468-RR (n=52) vs MDA-MB-468 cells (n=47). Data represented as mean ± SD, and statistical analysis involved one-way ANOVA with Dunnett’s multiple comparisons tests. C. The mRNA expression of NRF2 and HNRNPL for the MDA-MB-468-RR and NRF2-knockdown cells (shNRF2–1 and shNRF2–2). n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests. D. The mRNA expression of NRF2 and HNRNPL for the 4T1-RR and NRF2-knockdown cells (shNRF2–1 and shNRF2–2). n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests. E. The mRNA expression of NRF2 and HNRNPL for the MDA-MB-468-RR cells treated with DMSO or ML385 (5uM) for 72 hours (n=3, mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). F. The mRNA expression of NRF2 and HNRNPL for the MDA-MB-468 cells treated with DMSO or DMF (5uM) for 24 hours (n=3, mean ± SD, unpaired t-test two tailed). G. The fold enrichment of NRF2 binding to the HNRNPL promoter region identified by ChIP-qPCR when pulling down NRF2 vs IgG (n=3, mean ± SD, unpaired t-test two tailed).

Fig 5: HNRNPL stabilizes ITGB3 mRNA and regulates ITGβ3 expression.(A) Gene sets from “REACTOME” up-regulated in the 4T1-RR and MDA-MB-468-RR cells with the red color signifying gene sets associated with RNA metabolism. (B and C) The transcript stability of ITGB3 was compared between the MDA-MB-468-RR and MDA-MB-468 parental cells (B) and the 4T1-RR and 4T1 parental cells (C) by quantifying ITGB3 mRNA after actinomycin D treatment (0, 1, or 2 hours). Data were normalized to the expression at the 0-hour time point (n = 3, means ± SD, two-tailed, unpaired t test). (D and E) Comparison of mRNA and protein expression of HNRNPL in parental and radioresistant MDA-MB-468 (D) and 4T1 (E) cells (n = 3, means ± SD, two-tailed, unpaired t test). (F) MDA-MB-468-RR control (shCtrl) and HNRNPL knockdown cells (shHL1 and shHL2) were compared for HNRNPL expression by immunoblotting (left panel) and the expression of ITGB3 mRNA (right panel) (n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). (G) The stability of ITGB3 mRNA was determined by qPCR after 0, 1, 2, and 4 hours of actinomycin D treatment (n = 3, means ± SD, two-way ANOVA with Tukey’s multiple comparisons tests). (H) 4T1-RR control (shCtl) and HNRNPL knockdown cells (shHL1 and shHL2) were compared for HNRNPL expression by immunoblotting (left panel) and the expression of ITGB3 mRNA (right panel) (n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). (I) The stability of ITGB3 mRNA was determined by qPCR after 0, 1, 2, and 4 hours of actinomycin D treatment (n = 3, means ± SD, two-way ANOVA with Tukey’s multiple comparisons tests). (J) Surface expression of ITGβ3 in MDA-MB-468-RR cells and HNRNPL knockdown cells (n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests). (K) Surface expression of ITGβ3 in 4T1-RR cells and HNRNPL knockdown cells (n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons tests).