Journal: HemaSphere
Article Title: A microRNA expression signature in infant t(4;11) KMT2A::AFF1+ BCP‐ALL uncovers novel therapeutic targets
doi: 10.1002/hem3.70353
Figure Lengend Snippet: MiR‐194, miR‐99b, and miR‐125a‐5p act through their downstream targets CA5B, PPP3CA, and PPP2R5C. (A) CA5B, PPP3CA, and PPP2R5C are predicted targets of miR‐194, miR‐99b, and miR‐125a‐5p, respectively. (B) CA5B , (C) PPP3CA, and (D) PPP2R5C expression in leukemic blasts, non‐blasts from KMT2A::AFF1+ B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) patients, and normal FL pro‐B lymphoid cells. Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) of (E) CA5B , (F) PPP3CA, and (G) PPP2R5C in SEM cells that overexpress miRVANA® mimics ( n ≥ 3). (H) Western blots of SEM cells transfected with miRVANA® mimics for CA5B, PPP3CA, and PPP2R5C. The relative quantification (RQ) according to the control condition is presented. β‐Actin was used as a reference gene. (I) Luciferase assay in 293T cells using pGL3‐promoter‐UTR vectors of CA5B, PPP3CA, and PPP2R5C to assess microRNA‐mediated regulation ( n ≥ 3). (J) Proliferation and (K) apoptosis of SEM CRISPR‐Cas9 cells knocked out with CA5B, PPP3CA, and PPP2R5C ( n ≥ 3). Data are presented as mean ± SEM and compared using a Mann–Whitney U test with a bilateral P‐value according to the indicated reference value (REF): P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).
Article Snippet: The primary antibody was added at a dilution of 1:1000 in 5% milk TBST Buffer and incubated overnight at 4°C, with mild shaking: human Carbonic Anhydrase VB/CA5B antibody (Biotechne Cat# AF3176), rabbit anti‐PPP2R5C antibody affinity purified (Bethyl Laboratories Inc Cat# A303‐814A), PPP3CA polyclonal antibody (Elabscience Cat# E‐AB‐14813), and monoclonal anti‐β‐actin antibody produced in mouse (Sigma Cat# A2228).
Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Quantitative Proteomics, Control, Luciferase, CRISPR, MANN-WHITNEY