Additional file 1: Table S1. of Oct4 transcriptionally regulates the expression of long non-coding RNAs NEAT1 and MALAT1 to promote lung cancer progression

Supplementary methods. Anchorage-independent growth assay, Tumor-sphere formation assay, Tumor formation assay, Western blot analysis. Table S1. The plasmids and their characteristics used in the current study. Table S2. The ChIP-PCR primers used in the current study. Table S3. The cDNA primers used in the current study. Table S4. The construction primers of promoters and enhancers used in the current study. Figure S1. Oct4 promoted lung cancer tumorigenesis in vitro and in vivo. A Anchorage-independent assays in empty vector stably-transfected cell line (vector) and two biological replicates of Oct4 stably-overexpressed A549 and CL1-0 cell lines (Oct4#1, Oct4#2). Results were photographed (left) and quantified (right). B Transwell migration and invasion assay analysis of stably-transfected cell lines in A549 and CL1-0 cells. Results were photographed (left) and quantified (right). P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. C In vitro tumor sphere formation assay of A549 lung cancer cells stably expressing Oct4#1 or vector photographed (top) and quantified (middle). In vivo tumor formation assay using limited cell number (100, 1000, and 5000 cells) of vector and Oct4#1 cells. Tumor incidence of mice was analyzed at 8 weeks after implantation. D The immunoblots (upper) and qRT-PCR (lower) confirmed Oct4 expression in A549 and CL1–0 stable clones. Figure S2. Expression of lncRNAs in CL1–0 lung cancer cells manipulated for Oct4. A, B qRT-PCR analysis of eight lncRNAs expression in CL1-0 cells stably overexpressing Oct4 (Oct4#1, Oct4#2) (A) or Oct4-silenced CL1-0 cells (si-Oct4#1, si-Oct4#2) (B). Target lncRNA expression levels were normalized to GAPDH expression levels. Data represent mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S3. Oct4 positively regulated NEAT1 and UCA1 lncRNAs transcription in normal bronchial epithelial BEAS-2B cells. qRT-PCR analysis of selected lncRNAs expressions in BEAS-2B cells overexpressing Oct4. Target lncRNA expression levels were normalized to GAPDH expression levels. Data represent mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S4. RNA expression level of the manipulated NEAT1 and MALAT1 in A549 lung cancer cells. A549 cells transfected with NEAT1 expression vector (A) or MALAT1 expression vector (B), si-NEAT1 oligo (si-NEAT1) (C) or si-MALAT1 oligo (si-MALAT1) (D) were harvested and subjected to qRT-PCR assays for NEAT1 and MALAT1 RNA expression. Data are mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; ***P < 0.001. Figure S5. RNA and protein expression level of the manipulated Oct4, NEAT1 and MALAT1 in A549 lung cancer cells. A549 cells were transfected with expression vectors of NEAT1 (A) or MALAT1 (B) alone or together with si-Oct4 oligo (si-Oct4). A549 cells were transfected with si-NEAT1 oligo (si-NEAT1) (C) or si-MALAT1 oligo (si-MALAT1) (D) alone or together with Oct4 expression vector. Cell lysates were subjected to qRT-PCR assays for Oct4, NEAT1 and MALAT1 RNA expression or Western blot analysis for Oct4 protein expression (inset). GAPDH serves as an internal control. Data are mean ± SEM. P-values were determined by two-way ANOVA. **P < 0.01; ***P < 0.001. (PDF 911 kb

Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.info:eu-repo/semantics/publishe