Journal: Cancer research
Article Title: Targeting of CD38 by the tumor suppressor miR-26a serves as a novel potential therapeutic agent in multiple myeloma
doi: 10.1158/0008-5472.CAN-19-1077
Figure Lengend Snippet: (A) Human naïve B cells were isolated from mormal donor peripheral blood, and the purity was deteremined by flowcytometry. (B) The naïve B cells were labeled by CFSE, cultured in 24-well plates and stimulated for 3 days with 2.6 μg/ml F(ab’)2 fragment goat anti-human IgA+IgG+IgM (H+L), 100 ng/ml recombinant human soluble CD40L, 1.0 mg/ml CpG oligodeoxynucleotide 2006, and 50 U/ml recombinant IL-2. Day 3 activated B cells were washed and cultured up to 4 days with 50 U/ml IL-2, 50ng/ml IL-6, 50 ng/ml IL-10, and 2 ng/ml IL-12 and then followed by a 3 days culture with 50ng/ml IL-6, 10ng/ml IL-15 and 500U/ml IFN-α. Cell surface CD38 and CD138 expression was determined by flowcytometry. (C) qRT-PCR was used to determine the expression of miR-26a. (D) The CD38 signal in human tonsil tissue samples was detected by immunohistochemistry, and the miR-26a signal was examined by in situ hybridization. Area 1: Germinal center B cells. Area 2: plasma cells. Scale bar = 100 μM. (E) BLIMP1 mRNA level in naïve B cells and day 10 differentiated B cells was examined by qRT-PCR. Naïve B cells were activated for 3 days and transfected with one of the BLIMP1 siRNAs, with seqeunce scrambled siRNA as control. The cells were differentiated for another 7 days and the cell surface CD38 expression was determined by flowcytometry (F) BLIMP1 expression was determined by qRT-PCR and immunoblotting (G) and the miR-26a level was determined by qRT-PCR (H). Naïve B cells were activated for 3 days and then transfected with miR-control or miR-26a mimic. The cells were cultured with IL-2, IL-6, IL-12 and IL-10, allowed to differnentiate for 4 days and then followed by a 3 days culture with IL-6, IL-15 and IFN-α. Cell surface CD38 and CD138 expression was determined by flowcytometry (I). (J) Bone marrow biopsies were obtained from the same MM patient before daratumumab treatment and after became daratumumab resistant. The CD38 expression was examined by IHC and miR-26a level was examined by ISH. Scale bar = 100μM.
Article Snippet: Formalin-fixed paraffin-embedded sections were deparaffinized and then incubated with rabbit anti-CD38 polyclonal antibody (Cell Signaling Technology, #14637S) or rabbit anti-Ki-67 polyclonal antibody (Cell Signaling Technology, #9027S) or rabbit anti-cleaved caspase-3 monoblonal antibody (Cell Signaling Technology, #9664S) at 4°C overnight.
Techniques: Isolation, Labeling, Cell Culture, Recombinant, Expressing, Quantitative RT-PCR, Immunohistochemistry, In Situ Hybridization, Clinical Proteomics, Transfection, Control, Western Blot