Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: Mutant p53 and p63 bind to the let-7i promoter to regulate let-7i expression. (a) Schematic representation of let-7i promoter region containing the putative mutant p53-binding site. (b) H1299 cells stably expressing EV or p53R273H were cotransfected with the luciferase promoter plasmid carrying the let-7i promoter (pGL4-let-7i) and pRL-TK (internal control). Luciferase activity was measured after 36 h (left panel). Repression of let-7i promoter by mutant p53 was also analyzed by cotransfecting H1299-p53R273H cells with control (CTL) or p53 siRNAs and pGL4-let-7i for 48 h and measuring luciferase activity (right panel). (c) Parental H1299 cells were cotransfected with the let-7i promoter reporter (pGL4-let-7i), EV or p53R273H plasmids and pRL-TK (internal control). Luciferase activity was measured after 48 h. The effect of stable knockdown of mutant p53 in MDA-MB-231 and MIA-PaCa-2 cells on mature let-7i (d) and primary let-7i (e) levels was assessed by qRT–PCR normalized to U6 or UBC. (f) Mutant p53 co-immunoprecipitates with p63 and p73 in whole-cell lysates prepared from MDA-MB-231 cells. (g) Chromatin immunoprecipitation (ChIP) assays were performed to pulldown mutant p53 or p63 from MDA-MB-231 cells. Fold enrichment of the let-7i promoter region in the mutant p53 or p63 ChIP was determined by qPCR. (h) MDA-MB-231 cells were transfected with CTL or p63 siRNAs for 48 h and the levels of primary let-7i transcript (left panel) or mature let-7i (right panel) was assessed by qRT–PCR normalized to UBC or U6.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Mutagenesis, Expressing, Binding Assay, Stable Transfection, Luciferase, Plasmid Preparation, Control, Activity Assay, Knockdown, Quantitative RT-PCR, Chromatin Immunoprecipitation, Transfection

Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: A mutant p53–p63 complex regulates let-7i transcription through p63. (a) Schematic representation of the let-7i promoter region containing the putative mutant p53/p63-binding site (FL), lacking the mutant p53/p63 binding region (Δ400) or lacking the p63 response element (Δp63RE). (b) MDA-MB-231 cells were cotransfected with pGL4, pGL4-let-7i or pGL4-let-7i-Δ400 or pGL4-let-7i-Δp63RE and CTL, p53 or p63 siRNAs. Luciferase activity was measured after 48 h. (c) ChIP assays were performed to pulldown mutant p53 or p63 from MDA-MB-231 cells transfected with CTL, p53 or p63 siRNAs. Fold enrichment of let-7i promoter region in the mutant p53 or p63 ChIP was determined by qPCR. (d) p63 was immunoprecipitated (IP) from MDA-MB-231 whole-cell lysates in the presence or absence of DNase, and immunoblotting for p53 was performed. (e) Sequential ChIP assays were performed to HA-p63 and then mutant p53 from MDA-MB-231. Enrichment of let-7i promoter region in ChIP reactions was measured by qPCR. (f) Parental H1299 cells were transfected with HA-p63 after 48 h and immunoblotting was performed for using anti-HA. (g) Parental H1299 cells were cotransfected with the let-7i promoter (pGL4-let-7i-FL), EV or HA-p63 or HA-p63 and p53R273H plasmids and pRL-TK (internal control). Luciferase activity was measured after 48 h. Error bars represent mean±s.d. from three independent experiments. *P < 0.05; #P <0.01; **P< 0.005; ##P < 0.001.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Control

Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: let-7i inhibits the 3′ UTR of multiple oncogenes and RNA post-transcriptional modification genes. MDA-MB-231 cells were reverse-transfected with CTL or let-7i mimics for 36 h and changes in mRNA expression of known let-7 targets (a) and novel targets (b) identified in the microarrays were validated by qRT–PCR normalized to UBC. (c) MDA-MB-231 cells were reverse-transfected with CTL or let-7i mimics for 36 h and the protein levels of CPSF1 and MYC were analyzed by immunoblotting. β-actin was used as loading control. (d) Luciferase assays were performed from H1299-R273H cells cotransfected with psiCHECK2 (Vector), psiCHECK2 containing the 3′UTR of known let-7 targets (E2F5 or NRAS) or psiCHECK2 containing the 3′ UTR of nine RNA post-transcriptional modification genes and CTL or let-7i mimics. (e) Effect of silencing let-7i in H1299 cells with a let-7i sponge (let-7i-Spg) on the expression of let-7i target mRNAs was assessed by qRT–PCR. (f) Expression of CPSF1 and MYC protein from H1299 stably transfected with CTL-spg or let-7i sponge (let-7i-Spg) was measured by immunoblotting. β-actin was used as loading control. (g) mRNA levels of E2F5, LIN28B, MYC and NRAS were measured in breast cancer patient samples that were wild-type for p53 (WTp53) or had a missense mutation in p53 (MTp53) by qRT–PCR relative to GAPDH. (h) Schematic model suggesting a mechanism by which mutant p53 drives cell cycle progression, migration and invasion. Mutant p53 (MTp53) inhibits let-7i expression by inhibiting p63 at the let-7i promoter to upregulate let-7i targets including NRAS, MYC, E2F5, LIN28B, DDX18 and CPSF1 to enhance cellular proliferation, migration and invasion. Error bars represent mean ±s.d. of three independent experiments. *P < 0.05; #P < 0.01; **P < 0.005; ##P < 0.001.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Modification, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Luciferase, Plasmid Preparation, Stable Transfection, Mutagenesis, Migration

Journal: Cell reports

Article Title: Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD + /SIRT6 axis

doi: 10.1016/j.celrep.2021.109917

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-PCNA (Immunoblot-1:2500) , Santa Cruz Biotechnology , Cat# sc-56.

Techniques: Western Blot, Immunofluorescence, Recombinant, Virus, Protease Inhibitor, DC Protein Assay, Staining, CRISPR, Transfection, Mutagenesis, Bioassay, Modification, Cloning, Control, Binding Assay, Expressing, Plasmid Preparation, Software, Flow Cytometry

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 1 KCNAB2 is downregulated in human NSCLC. GEO databases (GEO44077, GEO33532) shows the KCNAB2 mRNA transcripts in human NSCLC tissues and normal lung tissues (A, B). Kaplan Meier survival analyses of KCNAB2-low and KCNAB2-high NSCLC patients in GEO dataset (GSE3141) was presented (C). ROC curve was used to evaluate the predictive value of the KCNAB2 in lung adenocarcinoma (D) and lung squamous cell carcinoma (E). Calibration model was used to evaluate the value of KCNAB2 in predicting the prognosis of lung cancer patients (F). qRT-PCR and Western blotting were applied for testing the expression of KCNAB2 in NSCLC cells (A549 and H1299) and alveolar epithelial cells (HPAEPIC), with results quantified (G, H). KCNAB2 immunofluorescence images in the described cells were shown. Scale bar = 10 μm (I). The representative human tissue KCNAB2 IHC images were shown. Scale bar = 50 μm (J). A mean ± standard deviation (SD, n = 3) was used to represent the data. Results from three repeated experiments were obtained. *P < 0.05, ***P < 0.001.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 2 Exogenous overexpression of KCNAB2 inhibits NSCLC cell proliferation, migration and invasion. A549 and H1299 cells with KCNAB2 cDNA-expressing lentiviral construct (“KCNAB2-OE”) or empty vectors (“Vec”) were established. KCNAB2 expression among stable cells was measured via qRT-PCR (A) and Western blotting (B). Cell colony formation (C), proliferation (“EdU assay”, D), migration and invasion (“Transwell assays”, E and F), motility (phagokinetic track motility assays, (G) were examined. A mean ± standard deviation (SD, n = 3) was used to represent the data. Results from three repeated experiments were obtained. *P < 0.05, ***P < 0.001. Scale bar = 100 μm.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Over Expression, Migration, Expressing, Construct, Quantitative RT-PCR, Western Blot, EdU Assay, Standard Deviation

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 3 KCNAB2 overexpression provokes apoptosis in NSCLC cancer cells. The expression of listed proteins in the described cells was assessed via Western blotting assay (A), with the results quantified (A). Mitochondrial depolarization was detected by JC-1 staining assay (B). Cell apoptosis was detected by TUNEL staining (C) and Annexin V-PI FACS assays (D). A mean ± standard deviation (SD, n = 3) was used to represent the data. Results from three repeated experiments were obtained. “Clvd” stands for “cleaved”. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 100 μm.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Over Expression, Expressing, Western Blot, Staining, TUNEL Assay, Standard Deviation

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 4 KCNAB2-knockout promotes proliferation, migration as well as invasion of NSCLC cell. A549 and H1299 cells with CRISPR/Cas9- KCNAB2-KO construct (“KCNAB2-KO”) or empty vector (“Cas9-C”) were established. KCNAB2 expression among stable cells was measured via via qRT-PCR (A) and Western blotting assays (B). The cell colony formation (C), proliferation (D), migration and invasion (E and F), and motility (G) were evaluated by assays as described, with data quantified. A mean ± standard deviation (SD, n = 3) was used to represent the data. Results from three repeated experiments were obtained. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 100 μm.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Knock-Out, Migration, CRISPR, Construct, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 5 KCNAB2-driven inhibition on NSCLC cell progression is partly mediated by inactivating AKT-mTOR cascade. Differentially expressed proteins (A) and relative signaling pathways (B) were analyzed by protein chip analyses on KCNAB2-overexpressed NSCLC cells comparing vector control cells. The listed protein’s expression profile was measured (C, D), with the results quantified (C, D). Cell proliferation (“EdU assay”, E) and migration (“Transwell assay”, F) of KCNAB2-KO A549 cells treated with MK-2206 were detected. Cell proliferation, migration and invasion capacities were determined by conducting EdU-nuclei (G) and “Transwell” (H, I) assays on KCNAB2-OE A549 cells stably expressing constitutively active mutant Akt (S473D, “caAkt1”) or empty vector (“Vec”). Cell apoptosis was examined by TUNEL staining (J). A mean ± standard deviation (SD, n = 3) was used to represent the data. Results from three repeated experiments were obtained. *P < 0.05. Scale bar = 100 μm.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Inhibition, Protein-Protein interactions, Plasmid Preparation, Control, Expressing, EdU Assay, Migration, Transwell Assay, Stable Transfection, Mutagenesis, TUNEL Assay, Staining, Standard Deviation

Journal: Cell death discovery

Article Title: KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo.

doi: 10.1038/s41420-023-01679-5

Figure Lengend Snippet: Fig. 6 KCNAB2 overexpression inhibits NSCLC cell growth in vivo. The mice volumes (A) and body weights (B) were recorded every 10 days. Tumors were isolated (C, D) and weighed individually (E) after 40 days. Expression of KCNAB2 mRNA and proteins in tumor tissue lysates were measured via qRT-PCR (F) and Western blotting (G), with data quantified. The representative IHC images of KCNAB2 (H) and Ki67 (I) in tumor tissues were showed, Scale bar = 50 μm. Representative TUNEL staining images in tumor tissues (J), Scale bar = 100 μm. A mean ± standard deviation (SD) was used to represent the data. *P < 0.05, ***P < 0.001.

Article Snippet: The membranes were then blocked with skim milk (10%) and incubated with following indicated primary antibodies: KCNAB2 (#orb324737, biorbyt, 1:2500 dilution), Tubulin (#ab179513, Abcam, 1:5000 dilution), anti-Akt (#4060, Cell Signaling Technology, 1:3500 dilution), antiS6 (#2217, Cell Signaling Technology, 1:3500 dilution), anti-phospho-S6 (#ab12864, Abcam, 1:3500 dilution), anti-phospho-Akt (S473) (#4060, Cell Signaling Technology, 1:3500 dilution), anti-cleaved-poly (ADP-ribose) polymerase (PARP) E51 (#ab32064, Abcam, 1:1500 dilution), anti-cleavedCaspase 9 (#ab2324, Abcam, 1:1500 dilution) overnight at 4 °C.

Techniques: Over Expression, In Vivo, Isolation, Expressing, Quantitative RT-PCR, Western Blot, TUNEL Assay, Staining, Standard Deviation