Journal: bioRxiv

Article Title: Notch1 regulates breast cancer stem cell function via a non-canonical cleavage-independent pathway

doi: 10.1101/2020.02.28.970764

Figure Lengend Snippet: Immunoblot analysis of: (A) Tsp-1, c-myc and actin expression in BPE cells in the presence and absence of ectopic expression of SV40 Large T antigen (LT) (BPLE), and HRasV12 (BPLER); (B) Myc, p53 and actin expression in wild type BPE cells transduced with vector controls pLKO.1-puro (V1), pMKO-neo (V2), pLKO.1shp53 (sh1) and pMKOneoshp53 (sh2); (C) Myc, p53 and actin expression in normal HME, normal human bronchial epithelial cells (NHBE) transduced with vector controls pLKO.1-puro (V1), pMKO-neo (V2), pLKO.1shp53 (sh1) and pMKOneoshp53 (sh2); (D) Myc, p53 and actin expression in BPE1, HME, and NHBE cells that were transiently transfected with pCMVneo or pCMVneo-p53 (p53); (E) Myc, p53 and actin expression in HME and BPE3 cells that were untreated (-) or treated with 25μM Nutlin-3: (F) Myc, p53 and actin expression in BPE and HME cells that were transiently transfected with pCMVneo, pCMVneo-p53 (wt) or pCMVneo-p53R175H; (G) Growth curve of HME, BPE, MCF-7 and MDA-MB-231 (231) cells that were untreated (black line) or treated with 25μM Nutlin-3/day for 5 days (Grey line); (H) Western blot of Myc, p53 and actin expression in A549 lung cancer cells that were untreated (-) or treated with 25μM Nutlin-3; (I) Western blot of Myc, p53 and actin expression in LNCaP prostate cancer cells that were untreated (-) or treated with 25μM Nutlin-3; (J) Western blot of Myc, p53 and actin expression in HCT116 colon cancer cells that were untreated (-) or treated with 25μM Nutlin-3.

Article Snippet: Western blots were performed as previously described using the following antibodies: c-myc (rabbit pAb, Cell Signaling Technologies, Danvers, MA), p53 (rabbit pAb, Cell Signaling Technologies), p21 (mAb clone DCS60, Cell Signaling Technologies), GAPDH (rabbit pAb, Trevigen, Gaithersburg, MD), SIRT1 (rabbit mAb E104, Abcam, Cambridge, MA), phosphorylation-p53 (Cell Signaling Technologies), Ac-p53 (rabbit pAb, Cell Signaling Technologies), Notch1 (rabbit mAb D1E11, Cell Signaling Technologies), β-actin (mouse mAb AC-15, Abcam), CD44 (Ab24504, Abcam), and MDM2 (PAB1729, Abnova, Taipei, Taiwan).

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Notch1 regulates breast cancer stem cell function via a non-canonical cleavage-independent pathway

doi: 10.1101/2020.02.28.970764

Figure Lengend Snippet: Immunoblot analysis of: (A) Acetylated p53 (Ac-p53), total p53 and actin expression in BPE and HME cells that were untreated (-) or treated with Nutlin-3 for 8 hours; (B) SIRT1 and actin expression in HME, BPE, lung MRC5 fibroblasts and hFhT cells; (C) Myc, acetylated p53 (Ac-p53) and actin expression in BPE cells transduced with vector alone (-), or wtSIRT1 (wt) that were untreated (-) or treated with 25μM Nutlin-3 for 8 hours (+); (D) Myc, acetylated p53 (Ac-p53) and actin expression in HME cells transduced with vector alone (-) or SIRT1-HY (HY) that were untreated (-) or treated with 25μM Nutlin-3 for 8 hours (+); (E) Notch1, NICD and actin expression in HME and BPE cells; (F) Notch1, NICD, SIRT1 and actin expression in wild type BPE and cells transduced with two Notch1 shRNAs; (G) Myc, Ac-p53, p53 and actin expression in BPE and BPE-shNotch cells that were untreated (-) or treated with Nutlin-3 (+) for 8 hours; (H) SIRT1 and actin expression in BPE cells that were untreated (-) or treated with 25µM DAPT (D) and15μM SAHM1 (S) for 24 hours; (I) pAKT, total Akt, and actin expression in BPE cells that were untreated (-) or treated with 15μM SAHM1 (+) for 1 hour; (J) (Left) SIRT1 and actin expression in BPE cells that were untreated (-) or treated with 25μM DAPT (D), 15μM SAHM1 (S), 10μM LY294002 (LY), 10µM MK2206 (MK), or 250nM Torin (T); ( Right) pAkt, total Akt and actin in BPE cells that were untreated (-) or treated with 100 or 250nM Torin; (K) Schematic diagram of Notch1 signaling pathway leading to the repression of SIRT1 expression.

Article Snippet: Western blots were performed as previously described using the following antibodies: c-myc (rabbit pAb, Cell Signaling Technologies, Danvers, MA), p53 (rabbit pAb, Cell Signaling Technologies), p21 (mAb clone DCS60, Cell Signaling Technologies), GAPDH (rabbit pAb, Trevigen, Gaithersburg, MD), SIRT1 (rabbit mAb E104, Abcam, Cambridge, MA), phosphorylation-p53 (Cell Signaling Technologies), Ac-p53 (rabbit pAb, Cell Signaling Technologies), Notch1 (rabbit mAb D1E11, Cell Signaling Technologies), β-actin (mouse mAb AC-15, Abcam), CD44 (Ab24504, Abcam), and MDM2 (PAB1729, Abnova, Taipei, Taiwan).

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: Notch1 regulates breast cancer stem cell function via a non-canonical cleavage-independent pathway

doi: 10.1101/2020.02.28.970764

Figure Lengend Snippet: Immunoblot analysis of: (A) CD44 and β-actin expression in BPE and HME cells that were untreated or treated with 25μM Nutlin-3; (B) CD44 and β-actin expression in BPE-shNotch1 cells that were untreated or treated with 25μM Nutlin-3; (C) CD44 and β-actin expression in HME cells that were untreated or treated with 25μM Nutlin-3 in the presence of 20μM SIRT1 inhibitor for 7 hours; (D) Notch1, NICD and β-actin expression in wild-type MDA-MB-231 (231) and BT-549 (549) breast cancer cells and 231 and 549 cells in which Notch1 was silenced via shRNA; (E) Acetylated p53 and β-actin expression in wild-type MDA-MB-231 (231) and BT-549 (549) breast cancer cells and 231 and 549 cells in which Notch1 was silenced via shRNA; (F) FACS analysis of the CD44 hi /CD24 lo populations in MDA-MB-231 breast cancer cells, in which Notch1 was silenced via shRNA (shNotch1), and treated with 20μM SIRT1 inhibitor (shNotch-SI) overnight prior to FACS sorting; (G) FACS analysis of the CD44 hi /CD24 lo populations in BT-549 breast cancer cells, in which Notch1 was silenced via shRNA (shNotch1), and treated with 20μM SIRT1 inhibitor (shNotch-SI) overnight prior to FACS sorting; (H) Light field microscopy of tumorspheres formed by MDA-MB-231 (231) and BT-549 (549) breast cancer cells, 231 and 549 cells in which Notch1 was silenced via shRNA, and 231- and 549shNotch1 cells treated with 20μM SIRT1 inhibitor daily for 12 days; (I) Plot of average area (μm 2 ) of tumorspheres formed by MDA-MB-231 (left) and BT-549 cells (right) depicted in .

Article Snippet: Western blots were performed as previously described using the following antibodies: c-myc (rabbit pAb, Cell Signaling Technologies, Danvers, MA), p53 (rabbit pAb, Cell Signaling Technologies), p21 (mAb clone DCS60, Cell Signaling Technologies), GAPDH (rabbit pAb, Trevigen, Gaithersburg, MD), SIRT1 (rabbit mAb E104, Abcam, Cambridge, MA), phosphorylation-p53 (Cell Signaling Technologies), Ac-p53 (rabbit pAb, Cell Signaling Technologies), Notch1 (rabbit mAb D1E11, Cell Signaling Technologies), β-actin (mouse mAb AC-15, Abcam), CD44 (Ab24504, Abcam), and MDM2 (PAB1729, Abnova, Taipei, Taiwan).

Techniques: Western Blot, Expressing, shRNA, Microscopy

Journal: bioRxiv

Article Title: Notch1 regulates breast cancer stem cell function via a non-canonical cleavage-independent pathway

doi: 10.1101/2020.02.28.970764

Figure Lengend Snippet: (A) Western blot of p73, p63, and β-actin in Notch1 hi and Notch1 low in SUM159 cancer stem cells; (B) RT-PCR of mRNA levels of TA and DN isoforms of p63 and p73 in Notch1 hi cells that were untreated or treated with SAHM1 and DAPT and Notch1 low cells that were untreated or treated with EX-527; (C) Western blot of p73, p63, acetylated p53 (Ac-p53), total p53 and β-actin in wild-type parental (P) SUM159 Notch1 hi cells and Notch1 hi cells in which the last 11 amino acids of the p53 protein had been deleted by CRISPR-Cas9 editing (Cr); (D) FACS analysis and bar graph of quantitation of apoptosis induced in SUM159 parental and CRISPR-edited, ΔAc-p53 cells, Notch1 hi and Notch1 low cells treated with 20nM paclitaxel; (E) Light-field images and bar graph of quantitation of tumorspheres formed by SUM159 parental and CRISPR-edited, ΔAc-p53 cells, Notch1 hi and Notch1 low cells; (F) Western blot of p73, p63, and β-actin in WT and shp63 SUM159 cells; (G) Western blot of p73, p63, and β-actin in WT and shp73 SUM159 cells; (H) FACS analysis and bar graph of quantitation of apoptosis induced in wild-type (parental) and shp73 SUM159 and cells, Notch1 hi and Notch1 low cells treated with 20nM paclitaxel; (I) Light-field images and bar graph of quantitation of tumorspheres formed by SUM159 parental and shp73, Notch1 hi and Notch1 low cells; (J) Western blot of TAp73 and β-actin in WT SUM159 and SUM159-TAp73 cells.

Article Snippet: Western blots were performed as previously described using the following antibodies: c-myc (rabbit pAb, Cell Signaling Technologies, Danvers, MA), p53 (rabbit pAb, Cell Signaling Technologies), p21 (mAb clone DCS60, Cell Signaling Technologies), GAPDH (rabbit pAb, Trevigen, Gaithersburg, MD), SIRT1 (rabbit mAb E104, Abcam, Cambridge, MA), phosphorylation-p53 (Cell Signaling Technologies), Ac-p53 (rabbit pAb, Cell Signaling Technologies), Notch1 (rabbit mAb D1E11, Cell Signaling Technologies), β-actin (mouse mAb AC-15, Abcam), CD44 (Ab24504, Abcam), and MDM2 (PAB1729, Abnova, Taipei, Taiwan).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, CRISPR, Quantitation Assay

Journal: Neural Regeneration Research

Article Title: Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis

doi: 10.4103/1673-5374.202946

Figure Lengend Snippet: Effects of STL on the protein expression of SIRT1, Ac-p53, Bcl-2 and Bax in the brain 24 hours after cerebral I/R. (A) Western blot assay for protein expression in each group. (B–E) Density analysis of SIRT1 (B), Ac-p53 (C), Bcl-2 (D), and Bax (E) protein expression. β-Actin was used as a loading control. The protein expressions were expressed as the optical density percentage of the target protein to β-actin. Values are expressed as the mean ± SEM ( n = 4 rats per group), and were analyzed by analysis of variance followed by the least significant difference test. ** P < 0.01, vs . sham group; ## P < 0.01, vs . I/R group; && P < 0.01, vs . I/R + STL (17.2 mL/kg) group. STL: Shuan-Tong-Ling ; I/R: ischemia/reperfusion; Ac-p53: acetylated-protein 53; EX527: SIRT1 inhibitor; SIRT1: silent information regulator 1.

Article Snippet: The membranes were incubated overnight with rabbit anti-mouse SIRT1 polyclonal antibody (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-mouse Ac-p53 polyclonal antibody (1:500; Santa Cruz Biotechnology, Buffalo, CA, USA), anti-mouse Bcl-2 monoclonal antibody (1:500; Santa Cruz Biotechnology), anti-mouse Bax monoclonal antibody (1:500; Santa Cruz Biotechnology) and goat anti-rat β-actin antibody (1:2,000; Boster, Wuhan, Hubei Province, China) at 4°C.

Techniques: Expressing, Western Blot, Control

Journal: Disease Models & Mechanisms

Article Title: Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice

doi: 10.1242/dmm.023366

Figure Lengend Snippet: Eµ-HDAC9 tumors display deregulated acetylation of BCL6 and p53. (A) IHC triple-immunostaining using the ABC-TSA method in mouse spleens from Eμ-HDAC9 and wild-type tumors, showing HDAC9 (red) expression in conjunction with levels of acetylated (Ac)-BCL6 (green). (B) Immunoblot analysis of Ac-p53 in Eµ- HDAC9 and wild-type spleen. GAPDH was used as a loading control. (C) ABC-TSA immunofluorescence analysis of Ac-p53 (green) and HDAC9 (red) in spleen in Eμ- HDAC9 and wild-type controls.

Article Snippet: Primary antibodies included polyclonal rabbit anti-Ac-p53 (Lys379) (Thermo Scientific), monoclonal rat anti-HDAC9 antibody (clone 45a7b5b), monoclonal mouse anti-FLAG M2 (Sigma), polyclonal rabbit anti-acetylated lysine (Millipore 06-933), polyclonal rabbit anti-BCL6 (ab19011), anti-CD45R(B220) (ab64100) and anti-CD3 (ab5690).

Techniques: Triple Immunostaining, Expressing, Western Blot, Control, Immunofluorescence