Fig. 2: Mutual repression of lineage-specific transcription factors (TFs) in the human SCLC cell line co-expression models.

a Immunoblots showing the expression profiles of the five intrinsic lineage-specific TFs in human SCLC cell lines. b Schema of the establishment of the co-expression models of lineage-specific TFs in SCLC cell lines. ASCL1-dominant (SCLC-A; Lu139 and H2107), NEUROD1-dominant (SCLC-N; H524 and H82), YAP1-dominant (SCLC-Y; SBC3), POU2F3-dominant (SCLC-P; H526), and ATOH1-dominant (HCC33) cell lines were used. Enhanced green fluorescent protein (EGFP)-expressing cells were used as controls. c Immunoblots showing the expression of lineage-specific TFs in Lu139^SCLC-A (left) and H524^SCLC-N (right) derivatives before and 5 days after doxycycline (Dox)-mediated induction of NEUROD1, YAP1^S127A, POU2F3, or ATOH1 for Lu139^SCLC-A, and of ASCL1, YAP1^S127A, POU2F3, or ATOH1 for H524^SCLC-N. The numbers below the ASCL1 and NEUROD1 blots indicate the values of the bands relative to the corresponding non-doxycycline control values after normalization against GAPDH. d Expression levels of endogenous ASCL1 transcripts after NEUROD1 co-expression in Lu139^SCLC-A cells (left) and of endogenous NEUROD1 transcripts after ASCL1 co-expression in H524^SCLC-N cells (right), relative to non-doxycycline controls after normalization against ACTB expression. The corresponding EGFP-expressing cells were used as controls. Cells were harvested after treatment with doxycycline for 72 hours. e Immunoblots showing changes in endogenous ASCL1 expression in Lu139^SCLC-A cells (top) and endogenous NEUROD1 expression in H524^SCLC-N cells (bottom) after incrementally increased co-expression of NEUROD1 and ASCL1, respectively. Cells were treated with doxycycline for 5 days. The numbers below the NEUROD1, ASCL1, and cleaved PARP blots indicate the values of the bands relative to the corresponding non-doxycycline control values after normalization against GAPDH. f Representative images of multiplex immunofluorescence staining of ASCL1 and NEUROD1 in Lu139^SCLC-A-TetO-NEUROD1 cells (top) and H524^4SCLC-N-TetO-ASCL1 cells (bottom) after treatment with different concentrations of doxycycline for 72 hours. Scale bar, 50 µm. g The expression level of ASCL1 mRNA relative to NEUROD1 mRNA in Lu165 cells. h Immunoblots showing the protein expression of ASCL1 and NEUROD1 in Lu165 cells. NEUROD1 is presented with short exposure (10 seconds) and long exposure (4 minutes) images. Lu139^SCLC-A cells and H2107^SCLC-A cells were used as positive controls for ASCL1 expression, while H524^SCLC-N cells and H82^SCLC-N cells were used as positive controls for NEUROD1 expression. i Immunoblots showing the expression of ASCL1 and NEUROD1 under ASCL1 or NEUROD1 knockdown in Lu165 cells. Cells were harvested 5 days after transfection of indicated siRNAs. The numbers below the ASCL1 and NEUROD1 blots indicate the values of the bands relative to the corresponding scrambled siRNA (siScr) control values after normalization against GAPDH. j CUT&RUN assay and quantitative PCR for binding sites of exogenous NEUROD1 on the ASCL1 loci in Lu139^SCLC-A-TetO-NEUROD1 cells (top). Lu139^SCLC-A-TetO-EGFP-expressing cells were used as a control (bottom). The anti-HA-tag antibody was used to detect exogenous NEUROD1 and EGFP with C-terminal HA-tags, and the rabbit IgG isotype control was used as a negative control. The amount of obtained DNA in each sample is represented as a signal relative to the total amount of input chromatin. k CUT&RUN assay and quantitative PCR for binding sites of exogenous ASCL1 on the NEUROD1 loci in H524^SCLC-N-TetO-ASCL1 cells (top). H524^SCLC-N-TetO-EGFP cells were used as a control (bottom). The anti-HA-tag antibody was used to detect exogenous ASCL1 and EGFP with C-terminal HA-tags, and the rabbit IgG isotype control was used as a negative control. The amount of obtained DNA in each sample is represented as a signal relative to the total amount of input chromatin. For CUT&RUN assays, cells were treated with doxycycline for 72 hours (j, k). Each Immunoblot image is representative of at least two independent procedures (a, c, e, h, i). Error bars indicate ± the standard error of the mean (SEM) from N = 3 independent experiments (d, g, j, k). Student’s t-test was used for two-group comparisons (d, j, k).