Fig 2: Effects of SLC7A11 and paclitaxel on the expression of related proteins analyzed by western blotting. The H-R-eGFP and H-R-SLC7A11 cells were treated with gradient concentration of paclitaxel for 72 hours. (A) Proteins were extracted and detected by western blotting to analyze the expression of p21 Waf1/Cip1, p27 Kip1, CDK2, CDK7, Cyclin A2, Cyclin B1 and Cyclin D3; (B) Expression of LC3 -II, LC3 -I, STX17, RAB33B, UVRAG, Atg7, Atg16L1 and Akt proteins were analyzed by western blotting. Representative blots were shown with GAPDH and β-Tubulin as loading control.

Fig 3: Inhibition of CDK1 and CDK7 decreases proliferation of TNBC cellssiRNA-mediated knockdown of CDK1 A. and B. and CDK7 C. and D. in MDA-MB-231-ERβ cells were verified at both the mRNA and protein level. The impact of CDK1 (B) and CDK7 (D) knockdown on the protein expression levels of their respective binding partners, cyclin B and cyclin H, as well as RNA Polymerase II phospho-serine 5 for siCDK7 treated cells, are shown. Total RNA Polymerase II and Vinculin are shown as loading controls. E. Proliferation rates of MDA-MB-231-ERβ cells six days after siRNA mediated knockdown of CDK1 and CDK7 in the absence (No Dox) and presence (Dox) of ERβ expression relative to scrambled siRNA control transfected cells. Dose response curves indicating the effects of dinaciclib F., a CDK1 inhibitor, and BS-181 G., a CDK7 inhibitor, on the proliferation rates of MDA-MB-231-ERβ cells in the presence and absence of ERβ expression after six days of treatment. The effects of four hours of drug mediated inhibition of CDK7 on the protein levels of total RNA polymerase II and RNA polymerase II phospho-serine 5 were also examined H. Vinculin is shown as a loading control. * Denotes significance at the P ≤ 0.05 level compared to controls.

Fig 4: CDK7 regulates the p38α/MYC pathway in NSCLC. a Changes of RNAPII C-terminal domain (CTD) phosphorylation and apoptosis markers in A549 and H460 cells after vehicle or THZ1 treatment at 48 h. b KEGG pathway analysis of differential expression genes for THZ1(200 nM, 24 h) treated cells over the vehicle in A549 cells. c KEGG pathway analysis of differential expression genes for THZ1 (200 nM, 24 h) treated cells over the vehicle in H292 cells. d Immunoblot analysis of p38α and MYC in A549 (left) and H460 (right) cells treated with vehicle or THZ1 at indicated time points. GAPDH was used as a loading control. e Immunoblot analysis of p38α, MYC, and CDK7 in A549 cells transfected with either scramble or CDK7 siRNAs. GAPDH was used as a loading control. f Immunoblot of MYC proteins after NSCLC cells transfected with p38α siRNAs or p38α vector. GAPDH was used as a loading control. g Immunoblot of MYC proteins in H1299, H1975, H23, and PC9 cells after THZ1 treatment with indicated dose at 48 h. h Luciferase reporter assays were performed to evaluate the transactivation potential of MYC in cells treated with THZ1 or transfected with p38α vector (n = 3) (*P < 0.05; ***P < 0.001). i Changes of MYC-targeted genes after THZ1 treatment in A549 and H292 cells by RNA-seq. j Correlation between CDK7 mRNA level and MYC mRNA level in TCGA NSCLC data (P < 0.0001). k Correlation between CDK7 protein level and MYC protein level in cohort I samples (P < 0.0001). l Correlation between CDK7 protein level and MYC protein level in cohort II samples (P < 0.0001). m Kaplan-Meier survival analysis of patients with different risk scores. High expression levels of CDK7, p38α, and MYC were defined as three risk factors. Patients were stratified into three risk groups with different survival outcomes as follows: low-risk group = none of the three were high expression (n = 12); medium-risk group = only one of the three was high expression (n = 32); high-risk group = at least two of the three were high expression (n = 33) (P = 0.038)

Fig 5: CDK7 inhibitor THZ1 promotes apoptosis and suppresses NSCLC growth in vitro and in vivo. a CCK8 assay depicting the changes in the viability of A549, H460, and SKMES1 cells treated with different doses of THZ1 for time points as indicated (n = 3). b Representative images showing colony formation of A549 and H460 cells treated with vehicle or THZ1. c Representative images of annexin V/propidium iodide staining showing increased apoptosis in NSCLC cells treated with THZ1. d Quantification of the annexin V positive cell ratio (n = 3) (***P < 0.001 as compared to vehicle group). e Representative images showing increased TUNEL positive cells (with nucleus stained in red) after THZ1 treatment at 48 h (× 40 magnification). f Quantification of the TUNEL positive cell ratio (n = 3) (***P < 0.001 as compared to vehicle group). g Caspase-3 activity was measured after NSCLC cells were treated with THZ1 (200 nM) for 48 h. Results are presented as fold-increase to vehicle-treated samples (n = 3) (*** P < 0.001). h Tumor growth curves of a lung adenocarcinoma PDX model treated with either vehicle or THZ1. Data represent mean ± SD (n = 6) (P = 0.0036). i Tumor growth curves of the H460 xenograft model treated with either vehicle or THZ1. Data represent mean ± SD (n = 8) (P < 0.0001). j H&E and IHC staining of Ki67 and cleaved caspase-3 in tumor tissue sections from H460 xenograft. Original magnification, × 400; scale bar, 20 μm