Figure 3. CSR and acceptor S region transcription are compromised by Med1 deficiency in primary B cells. (A, left) Percentage (+SD) of CSR relative to control cells from three to six independent experiments. The genotypes tested and number of mice were as follows: Med1F/FMb1Cre/+ (n = 37), Med1+/+ (n = 6), Mb1Cre/+ (n = 4), Med1F/F (n = 28), Med1F/+ (n = 11), or Med1F/+Mb1Cre/+ (n = 16). No difference between control genotypes (Med1+/+, Mb1Cre/+, Med1F/F, Med1F/+, and Med1F/+Mb1Cre/+) was observed. CSR in control cells was set to 100%. Statistical analysis was performed using Student’s t test. **, P ≤ 0.01; ***, P ≤ 0.0001. Right: CSR to IgE was evaluated by the levels of Iμ-Cε post-switch transcripts by RT-qPCR in control and Med1F/FMb1Cre/+ B cells cultured for 72 h with LPS + IL-4. Expression is normalized to Igβ and is presented relative to expression in control B cells (set as 1). Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. **, P ≤ 0.01. Data are representative of three experiments with two mice per genotype. (B) Representative example of surface expression of IgG1, IgG3, and CFSE dilution as determined by flow cytometry in Med1F/F and Med1F/FMb1Cre/+ B cells stimulated for 72 h with LPS + IL-4 or LPS alone. Percentage of switched cells is indicated. (C, top) RT-qPCR analysis for AID mRNA in control and Med1F/FMb1Cre/+ B cells cultured for 72 h with LPS + IL-4. Expression is normalized to Igβ and is presented relative to expression in control B cells (set as 1). Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. Data are representative of three experiments with two mice per genotype. Bottom: Western blot for β-actin and AID on whole-cell extracts from splenic B cells from Med1F/F and Med1F/FMb1Cre/+ mice cultured for 72 h with LPS and IL-4. Theoretical molecular masses in kilodaltons are indicated. Data are representative of three independent experiments. (D) RT-qPCR analysis for germline transcripts (GLT) at donor and acceptor S regions in Med1F/FMb1Cre/+ and control (Med1F/F) B cells cultured for 72 h with LPS alone or with LPS + IL-4 or LPS + IFN-γ. Expression is normalized to Igβ and is presented relative to expression in control B cells, set as 1. Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Data are representative of three independent experiments with two mice per genotype.
Figure 3.

CSR and acceptor S region transcription are compromised by Med1 deficiency in primary B cells. (A, left) Percentage (+SD) of CSR relative to control cells from three to six independent experiments. The genotypes tested and number of mice were as follows: Med1F/FMb1Cre/+ (n = 37), Med1+/+ (n = 6), Mb1Cre/+ (n = 4), Med1F/F (n = 28), Med1F/+ (n = 11), or Med1F/+Mb1Cre/+ (n = 16). No difference between control genotypes (Med1+/+, Mb1Cre/+, Med1F/F, Med1F/+, and Med1F/+Mb1Cre/+) was observed. CSR in control cells was set to 100%. Statistical analysis was performed using Student’s t test. **, P ≤ 0.01; ***, P ≤ 0.0001. Right: CSR to IgE was evaluated by the levels of Iμ-Cε post-switch transcripts by RT-qPCR in control and Med1F/FMb1Cre/+ B cells cultured for 72 h with LPS + IL-4. Expression is normalized to Igβ and is presented relative to expression in control B cells (set as 1). Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. **, P ≤ 0.01. Data are representative of three experiments with two mice per genotype. (B) Representative example of surface expression of IgG1, IgG3, and CFSE dilution as determined by flow cytometry in Med1F/F and Med1F/FMb1Cre/+ B cells stimulated for 72 h with LPS + IL-4 or LPS alone. Percentage of switched cells is indicated. (C, top) RT-qPCR analysis for AID mRNA in control and Med1F/FMb1Cre/+ B cells cultured for 72 h with LPS + IL-4. Expression is normalized to Igβ and is presented relative to expression in control B cells (set as 1). Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. Data are representative of three experiments with two mice per genotype. Bottom: Western blot for β-actin and AID on whole-cell extracts from splenic B cells from Med1F/F and Med1F/FMb1Cre/+ mice cultured for 72 h with LPS and IL-4. Theoretical molecular masses in kilodaltons are indicated. Data are representative of three independent experiments. (D) RT-qPCR analysis for germline transcripts (GLT) at donor and acceptor S regions in Med1F/FMb1Cre/+ and control (Med1F/F) B cells cultured for 72 h with LPS alone or with LPS + IL-4 or LPS + IFN-γ. Expression is normalized to Igβ and is presented relative to expression in control B cells, set as 1. Mean and SD of triplicate samples are shown. Statistical analysis was performed using two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Data are representative of three independent experiments with two mice per genotype.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal