Fig 2: miR-9-5p regulates the transcription factor Klf4 in HCC cells. RT-qPCR analysis of (A) Klf4 and (B) miR-9-5p in L02 and HCC cells. (C and D) RT-qPCR and (E) western blot analysis of mRNA and protein expression levels, respectively, of Klf4 and miR-9-5p at 48 h following transfection with 100 nM miR-9-5p mimic or 100 nM inhibitor, and their control groups in HCC cells. (F) The binding site of miR-9-5p was predicted in the Klf4 3'-UTR. Subsequently, the luciferase reporters of the WT or MUT miR-9-5p targeting Klf4 3'-UTR sites was constructed. Luciferase activity was measured 48 h following transfection of miR-9-5p mimic or negative control, together with WT or MUT Klf4 3'-UTR. *P<0.05; **P<0.01; ***P<0.001. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA; HCC, hepatocellular carcinoma; mimic NC, mimic negative control; inhibitor NC, inhibitor negative control; Klf4, Krüppel-like factor 4; 3'-UTR, 3'-untranslated region; WT, wild-type; MUT, mutation-type.

Fig 3: miR-9-5p overexpression in clinical samples is associated with low expression of Klf4 and a poor prognosis of HCC. Reverse transcription-quantitative polymerase chain reaction analysis of (A) Klf4 and (B) miR-9-5p in clinical tissue. (C) Inverse correlation between miR-9-5p and Klf4 in patients, as determined by Spearman's correlation. (D) miR-9-5p expression levels between the high and low expression groups, separated by the median value. A statistically significant difference existed between two groups. ***P<0.001. (E) Kaplan-Meier analysis of patients with HCC based on different expression levels of miR-9-5p obtained from The Cancer Genome Atlas HCC database (P=0.027). miR, microRNA; HCC, hepatocellular carcinoma; Klf4, Krüppel-like factor 4.

Fig 4: Higher levels of SOX2 confer PTX resistance in A549 non-small cell lung cancer cells. (A) mRNA expression levels of stemness-related genes were measured by reverse transcription-quantitative PCR in A549 and A549-PTX cells. (B) (Left) Protein expression levels of NANOG, KLF4, OCT4 and SOX2 were examined by western blotting in A549 and A549-PTX cells. (Right) Protein expression was semi-quantified using ImageJ. (C) Protein expression levels of SOX2 were examined by western blotting in A549-PTX cells transfected with siNC or SOX2 siRNA (siSOX2-1, siSOX2-2 and siSOX2-3). (D) Viability was examined using CCK-8 assays in A549-PTX cells transfected with siSOX2 or its control and treated with PTX for 48 h. (E) (Left) Protein expression levels of MDR1, ABCC10, ABCC2 and SOX2 were examined by western blotting in A549-PTX cells transfected with siSOX2 or its control. (Right) Protein expression was semi-quantified using ImageJ. (F) Protein expression levels of SOX2 were examined by western blotting in A549 cells overexpressing SOX2 or its vector control. (G) Viability was examined using CCK-8 assays in A549, A549-PTX and SOX2-overexpressing A549 cells treated with PTX for 48 h. (H) (Left) Protein expression levels of MDR1, ABCC10 and ABCC2 were examined by western blotting in A549 cells overexpressing SOX2 or its vector control. (Right) Protein expression was semi-quantified using ImageJ. GAPDH or Tubulin were used as a loading control in western blotting. All data are presented as the mean ± standard deviation. *P<0.05, ***P<0.001. ns, not significant. Relative, vs. respective control. SOX2, SRY-box transcription factor 2; PTX, paclitaxel; A549-PTX cells, PTX-resistant A549 non-small cell lung cancer cells; KLF4, Krüppel like factor 4; OCT4, octamer-binding transcription factor 4; siRNA/si, small interfering RNA; siNC, control siRNA; CCK-8, Cell Counting Kit-8; MDR1, multidrug resistance mutation 1; ABCC10, ATP binding cassette subfamily C member 10; ABCC2, ATP binding cassette subfamily C member 2; Ctl, control.

Fig 5: Pten activation by S380A, T382A, T383A mutations suppresses ESC pluripotency.a, b The domain structure of Pten in the “closed” conformation and “opened” conformation. c Western blots analysis showing the loss of phosphorylation in the Pten-A3 mutant ESCs. d Western blot analysis of WT, Pten-A3 mutant, and Pten-/- ESCs showing the expression of the pluripotency markers Nanog, Klf4, and Oct4. e Phosphorylation of Akt at S473 and T308 and phosphorylation of Gsk3ß at S9 by Western blot. f Scatter plots of transcript expression in WT and Pten-A3 mutant ESCs. Expression values are shown on a log10 scale. Yellow dots indicate upregulated genes in Pten-/- ESCs, and blue dots indicate downregulated genes (fold-change cutoff = 1.5, FDR threshold = 0.05). g Heat map of FPKM values of pluripotency genes in WT and Pten-A3 mutant ESCs. The heat map was normalized with sigma-normalization per row. See also Table S3. h A nude mouse injected with WT and Pten-A3 mutant ESCs on the left and right sides, respectively. i WT and Pten-A3 mutant ESCs were injected into immunodeficient mice and produced teratocarcinomas containing tissues representative of the three germ layers (mesoderm, ectoderm, and endoderm). j Teratomas from WT and Pten-A3 mutant cells generated at the contralateral positions aligned at the same positions in each genotype. k Weights (g) of teratomas from WT and Pten-A3 mutant cells.