1 / 6

Supplemental Figure 1

Supplemental Figure 1. A. B. Inhibitors concentration (µM).

shanae
Download Presentation

Supplemental Figure 1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Supplemental Figure 1 A B Inhibitors concentration (µM)

  2. Supplemental Figure 1: B cell survival (A) B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM) and ER-27319 (SYK, 50µM)) or with AMD3100 (100µM) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. One representative experiment from 4 is represented (average+SEM). (B) B cells were pre-treated with increasing concentrations of JNK and SYK inhibitors (SP600125 (JNK, 0,1µM to 20µM); ER-27319 and Piceatannol (SYK 1µM to 100µM)) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. Results from one representative experiment from two. Doted line represent the threshold of cell viability determined at 80% in comparison to Mock-NI condition.

  3. Supplemental Figure 2 # # # # # # # * * * * # * * * Supplemental Figure 2: Cell activation and inhibition of activation by kinases inhibitors. B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM); ER-27319 (SYK, 50µM); Piceatannol(SYK, 50µM)) and AMD3100 (100µM) for 4h before CD40L/IL-4 stimulation. 24h post-stimulation, iMFI of cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Average of iMFI + SEM of minimum 4 experiments for each conditions (4 to 6 different donors for the NA (non-activated) conditions and 6 to 10 donors for the CD40L/IL-4 conditions). NA; Non-activated (*=p<0,05 when comparing NT-NA condition with kinase inhibitor-NA and #=p<0,05 when comparing NT-CD40L/IL-4 condition and kinases inhibitor-CD40L/IL-4). NT: non-treated.

  4. Supplemental Figure 3 A * * * NT PDTC ER-27319 PD98059 SP600125 SB203580 GF109203 Piceatannol wortmannin B C # * # * #

  5. Supplemental Figure 3: AID mRNA expression in CD40L/IL-4 treated B cells. (A) B cells were pre-treated with kinase inhibitors; GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM), ER-27319 (SYK, 50µM) or Piceatannol (SYK, 50µM) for 4h before CD40L/IL-4 stimulation or cells were kept non-activated as control. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to non treated (NT) condition that was considered a fold change of 1. Each black (Non-activated conditions) or grey (CD40L/IL-4 stimulated conditions) plot represents one donor. Black bar represents the average of AID mRNA fold change . (B) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4 for 24h. iMFIof cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Fold changes were calculated in comparison to NT-NA (Non-Activated) condition that is considered a fold change of 1. Average of iMFI + SEM of minimum 4 independent experiments for each conditions (4 or 5 different donors regarding the condition). #=p<0,05 when comparing HIV condition with HIV/CD40L/IL-4 and *=p<0,05 when comparing CD40L/IL-4 condition with HIV/CD40L/IL-4(C) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to NT-NA condition that is considered a fold change of 1. Average + standard deviation of 2 independent experiments is shown.

  6. Supplemental Figure 4 V(D)J µ γ2 δ µ δ IgM GermlineIH-CH transcription CSR AID V(D)J γ2 IgG2 Excised DNA segment=circle transcript Productive (VDJ-CH) Ig transcript Supplemental Figure 4: Class Switch Recombination (CSR). Mechanism of class switch recombination that allows isotypeselection in mature activated B cells. This mechanism produces a DNA recombinant of the constant region of the immunoglobulin heavy chain and an excised DNA segment.

More Related