pslik idh2 r172k flag  (Addgene inc)

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: IDH mutations activate CEBPα-VDR-RXRα axis through 2HG production. ( A ) RT-qPCR showing gene expressions of VDR and RARA in HL60 IDH1 WT (clone 2: ○; clone 7: ☐) versus HL60 IDH1 R132H (clone 5: ◇; clone 11: △). ( B ) Western blot showing protein levels of VDR, RXRα and RARα in HL60 IDH1 WT (clones 2 and 7) versus HL60 IDH1 R132H (clones 5, 11 and 3) (left) and in HL60 IDH2 WT versus HL60 IDH2 R172K (right). ( C ) RT-qPCR showing gene expressions of VDR and RARA in 2HG-treated HL60 (200 µM, 1 week). ( D ) Western blot showing protein levels of VDR RXRα and RARα in 2HG-treated HL60 IDH1 WT (clones 2 and 7) (200 µM, 1 week). ( E ) RT-qPCR showing gene expression of and VDR and RARA in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) and IDH1 R132H (clone 5: ◇, clone 11: △) after CEBPA-KD by shRNA ( F ) Western blot showing protein levels of CEBPα, VDR, RARα and VDR in HL60 IDH1 WT (clones 2 and 7) and IDH1 R132H (clone 5 and 11) after CEBPA-KD by shRNA. ( G ) Western blot showing protein levels of VDR in 2HG-treated HL60 IDH1 WT (clone 2 and 7) with CEBPA-KD by shRNA (200 µM, 1 week). The uncropped western blot figures are presented in . * p < 0.05; ** p < 0.01.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Quantitative RT-PCR, Western Blot, Clone Assay, Expressing, shRNA

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: Combine treatment with VD and ATRA produces a synergistic induction of differentiation in a CEBPα-dependent manner. ( A ) HeatMap of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 WT versus HL60 IDH1 R132H and HL60 IDH2 WT versus HL60 IDH2 R172K treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( B ) Synergy Map of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 R132H-5 (left) and in HL60 IDH1 R132H-11 (right) treated for 3 days with 0.25–4 µM of ATRA and 25–400 nM of VD alone or in combination. ( C ) Combination index of differentiation (CD11b expression induction) in HL60 IDH1 R132H-5 and in HL60 IDH1 R132H-11 treated for 3 days with ATRA and VD in combination with constant ratio of each drug. ( D ) Morphological characterization by MGG staining of HL60 IDH1 WT-2 versus HL60 IDH1 R132H-11 treated for 5 days with ATRA 1 µM and VD 100 nM alone or in combination. ( E ) HeatMap of CD11b expression and CD15 expression (MFI, ratio to untreated) measured by flow cytometry in 2HG-treated (100(U937)-200 µM for 1wk) HL60 IDH1 WT-2 , HL60 IDH1 WT-4 , HL60 IDH1 WT-7 , HL60, U937, THP1 and KG1a treated for 3 days with ATRA (0.1 µM for U937, 1 µM for others) or VD (100 nM) alone or in combination. ( F ) CD11b expression (MFI) measured by flow cytometry in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (left panel) and in HL60 IDH1 R132H (clone 5: ◇, clone 11: △) shCTL vs. shCEBPα (right panel) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( G ) CD11b expression (MFI) measured by flow cytometry in 2HG-treated HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (2HG 200 µM for 3 days) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. * p < 0.05; ** p < 0.01, *** p < 0.005.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: IDH mutations activate CEBPα-VDR-RXRα axis through 2HG production. ( A ) RT-qPCR showing gene expressions of VDR and RARA in HL60 IDH1 WT (clone 2: ○; clone 7: ☐) versus HL60 IDH1 R132H (clone 5: ◇; clone 11: △). ( B ) Western blot showing protein levels of VDR, RXRα and RARα in HL60 IDH1 WT (clones 2 and 7) versus HL60 IDH1 R132H (clones 5, 11 and 3) (left) and in HL60 IDH2 WT versus HL60 IDH2 R172K (right). ( C ) RT-qPCR showing gene expressions of VDR and RARA in 2HG-treated HL60 (200 µM, 1 week). ( D ) Western blot showing protein levels of VDR RXRα and RARα in 2HG-treated HL60 IDH1 WT (clones 2 and 7) (200 µM, 1 week). ( E ) RT-qPCR showing gene expression of and VDR and RARA in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) and IDH1 R132H (clone 5: ◇, clone 11: △) after CEBPA-KD by shRNA ( F ) Western blot showing protein levels of CEBPα, VDR, RARα and VDR in HL60 IDH1 WT (clones 2 and 7) and IDH1 R132H (clone 5 and 11) after CEBPA-KD by shRNA. ( G ) Western blot showing protein levels of VDR in 2HG-treated HL60 IDH1 WT (clone 2 and 7) with CEBPA-KD by shRNA (200 µM, 1 week). The uncropped western blot figures are presented in . * p < 0.05; ** p < 0.01.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Quantitative RT-PCR, Western Blot, Clone Assay, Expressing, shRNA

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: Combine treatment with VD and ATRA produces a synergistic induction of differentiation in a CEBPα-dependent manner. ( A ) HeatMap of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 WT versus HL60 IDH1 R132H and HL60 IDH2 WT versus HL60 IDH2 R172K treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( B ) Synergy Map of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 R132H-5 (left) and in HL60 IDH1 R132H-11 (right) treated for 3 days with 0.25–4 µM of ATRA and 25–400 nM of VD alone or in combination. ( C ) Combination index of differentiation (CD11b expression induction) in HL60 IDH1 R132H-5 and in HL60 IDH1 R132H-11 treated for 3 days with ATRA and VD in combination with constant ratio of each drug. ( D ) Morphological characterization by MGG staining of HL60 IDH1 WT-2 versus HL60 IDH1 R132H-11 treated for 5 days with ATRA 1 µM and VD 100 nM alone or in combination. ( E ) HeatMap of CD11b expression and CD15 expression (MFI, ratio to untreated) measured by flow cytometry in 2HG-treated (100(U937)-200 µM for 1wk) HL60 IDH1 WT-2 , HL60 IDH1 WT-4 , HL60 IDH1 WT-7 , HL60, U937, THP1 and KG1a treated for 3 days with ATRA (0.1 µM for U937, 1 µM for others) or VD (100 nM) alone or in combination. ( F ) CD11b expression (MFI) measured by flow cytometry in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (left panel) and in HL60 IDH1 R132H (clone 5: ◇, clone 11: △) shCTL vs. shCEBPα (right panel) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( G ) CD11b expression (MFI) measured by flow cytometry in 2HG-treated HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (2HG 200 µM for 3 days) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. * p < 0.05; ** p < 0.01, *** p < 0.005.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Expressing, Flow Cytometry, Staining