Journal: Cancer Immunology, Immunotherapy

Article Title: Support of BCP-ALL-cells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis

doi: 10.1007/s00262-020-02835-x

Figure Lengend Snippet: IL-13, TGFβ and CD40L mediate Th-cell-induced AICDA expression in BCP-ALL-cells. a Primary BCP-ALL-cells were stimulated with CIT ± IFNγ for 3d. AICDA expression was determined using qRT-PCR after preamplification of target genes. Shown are mean ± SD of dCT values normalized to the geometric mean of HMBS , TBP , GUSB and YWAHZ . Different symbols represent different patients. p values were calculated using a mixed-effect analysis with Sidak correction for multiple comparison. p > 0.05 not significant (n.s.), p < 0.01** b Xenograft ALL671 BMMCs were stimulated using CIT ± IFNγ for 5d. Total AID protein expression was assessed using western blotting. β-actin was used as loading control. Western blot of primary cells was performed once. c RS4;11, TOM1 and SD1 cells were stimulated for 3d using different combinations of CD40L, IL-13 and TGFβ. AICDA expression was determined using qRT-PCR. Shown are mean ± SD of ddCT values normalized to the geometric mean of HMBS and GUSB and to unstimulated cells. Dots represent different replicates. d p values in the table were calculated from dCT values using three-way ANOVA with Geisser-Greenhouse correction for sphericity. e SD1 cells were stimulated using CD40L and IL-13 for 5d. AID protein expression was analyzed using western blotting. α-tubulin was used as loading control. Shown is a representative result of five replicates

Article Snippet: Stimulation with HA-tagged recombinant human CD40 Ligand (CD40L) (500 ng/ml) was complemented with anti-HA antibody (200 ng/ml, both R&D Systems) enabling multimer formation.

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Cancer Immunology, Immunotherapy

Article Title: Support of BCP-ALL-cells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis

doi: 10.1007/s00262-020-02835-x

Figure Lengend Snippet: Th-cell-induced AICDA is transcriptionally regulated via canonical NF-κB, Stat6 and Smad2/3 signaling in BCP-ALL-cells. a , b RS4;11 and SD1 cell lines were stimulated for 2 h with CD40L, IL-13 and TGFβ. a Localization of transcription factors in cytoplasmic and nuclear fraction was analyzed using western blotting. α-Tubulin and Lamin A/C were used as a loading and fractionation controls. RS4;11 cells do not express Lamin A/C. b Phosphorylation of Stat6 was analyzed using western blotting. β-actin was used as a loading control. c Transcription factors were knocked-down in RS4;11 cells by electroporation of siRNA for 48 h and stimulated with CD40L, IL-13 or TGFβ for 24 h. AICDA expression was analyzed using qRT-PCR. Shown are ddCT normalized to HMBS and siCtrl/unstimulated condition. p values were calculated using a mixed-effect analysis by comparing siCtrl and with targeting siRNA with same stimulation. Sidak correction for multiple comparison was used. p > 0.05 not significant (n.s.), p < 0.05*, p < 0.01**, n.a. not analyzed

Article Snippet: Stimulation with HA-tagged recombinant human CD40 Ligand (CD40L) (500 ng/ml) was complemented with anti-HA antibody (200 ng/ml, both R&D Systems) enabling multimer formation.

Techniques: Western Blot, Fractionation, Electroporation, Expressing, Quantitative RT-PCR, Comparison