cloning reactions  (Addgene inc)

Journal: Blood Advances

Article Title: Loss of p 53 enhances the tumor-initiating potential and drug resistance of clonogenic multiple myeloma cells

doi: 10.1182/bloodadvances.2022009387

Figure Lengend Snippet: Loss of p 53 enhances the tumor-initiating potential of MM cells. (A) Growth curves of p 53-wt (Scr) and p 53-KO MM.1S, MM.1R, MOLP8, NCI-H929, and KMS11 cells (n = 3). (B) Primary (1°) and secondary (2°) colony formation of p 53-wt (Scr) and p 53-KO MM.1S, MOLP8, and NCI-H929 cells (n = 5). (C) Colony formation of p 53-wt (Scr) and p 53-KO MM.1R cells (n = 3). (D) Colony formation of p 53-null KMS11 (Ctrl) and KMS11 p 53-overexpression cells (n = 3). (E) Frequency of CD138 neg and ALDH + cells within Scr and p 53-KO MM.1S, MM.1R, and MOLP8 cell lines by flow cytometry (n = 6).

Article Snippet: For Notch intracellular domain (NICD) overexpression, the NICD fragment (from TetO-FUW-NICD; Addgene) was cloned into the pINDUCER21 (Addgene) lentiviral vector, then transfected into MM.1S p 53-KO cells.

Techniques: Over Expression, Flow Cytometry

Journal: Blood Advances

Article Title: Loss of p 53 enhances the tumor-initiating potential and drug resistance of clonogenic multiple myeloma cells

doi: 10.1182/bloodadvances.2022009387

Figure Lengend Snippet: Notch1-Hes1 signaling is required for increased clonogenic growth and drug resistance of p 53-KO MM TICs. (A) Colony formation of ID1-KD, ID2-KD, and nontargeting scramble (Ctrl)-treated p 53-wt (Scr) and p 53-KO MM.1S cells. (B) Western blot analysis of NOTCH1 and HES1 expression in p 53-wt (Scr) and p 53-KO MM.1S cells after treatment with a GSI. (C) Colony formation of HES1-KD and nontargeting scramble (Ctrl)-treated p 53-wt (Scr) and p 53-KO MM.1S cells (n = 4). (D) Colony formation of GSI-treated p 53-wt (Scr) and p 53-KO MM.1S, MM.1R, and MOLP8 cells (n = 4). (E) Western blot analysis of HES1 expression after doxycycline-induced Notch1 intracellular domain overexpression in p 53-KO MM.1S cells. (F) Colony formation of Notch1 intracellular domain overexpressing p 53-KO MM.1S cells after GSI treatment (n = 6). (G) Colony formation of GSI-treated clinical MM non-del17p samples (n = 6) and del17p samples (n = 3). (H) Colony formation of p 53-KO MM.1S cells after treatment with GSI (2.5 nM) and melphalan (Mel, 1 uM) or GSI and bortezomib (BTZ, 1.5 nM) (n = 5). (I) Melphalan IC 50 values of GSI-treated p 53-wt (Scr) and p 53-KO MM.1S cells (n = 3).

Article Snippet: For Notch intracellular domain (NICD) overexpression, the NICD fragment (from TetO-FUW-NICD; Addgene) was cloned into the pINDUCER21 (Addgene) lentiviral vector, then transfected into MM.1S p 53-KO cells.

Techniques: Western Blot, Expressing, Over Expression