Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme promotes plasma cell (PC) formation in response to T-dependent and T-independent activating signals. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry analysis at 72 h. (B) Quantitation of the frequency of CD138 + PCs from (A). (C) Naïve B cells were stimulated with CD40L, IL-4, and IL-5 signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at 48, 72, and 96 h. (D) Quantitation of the frequency of CD138 + PCs from (C). (E) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at the indicated time points. (F) Quantitation of the frequency of CD138 + PCs from (E). Data represent the combination of at least 2 independent experiments with at least 3 mice each. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Article Snippet: B cells were stimulated with either 20 μg/mL LPS (L2630; Sigma), 20 ng/mL IL-2 (575406; BioLegend), and 5 ng/mL IL-5 (581504; BioLegend) or 500 ng/mL CD40L (R&D Systems, 8230-CL), 10 ng/mL IL-4 (R&D Systems, 404-ML), and 10 ng/mL IL-5.

Techniques: Clinical Proteomics, Ex Vivo, Concentration Assay, Flow Cytometry, Quantitation Assay, Control

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme induces more proliferating cells at later divisions. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h for all B cells in culture. (B) Quantitation of the mean division number (MDN) for all cultures in (A). (C) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, IL-5) signals ex vivo and 60 µM of heme or vehicle control added after 24 h. Representative flow cytometry histograms showing CTY dilution at the indicated time points for all B cells in culture. (D) Quantitation of the MDN of cell cultures in (C). (E) Representative flow cytometry analysis of CD138 + cells vs CTY dilution at the indicated time points. (F) Quantitation of the percentage of CD138 + cells within each division from (E). (G) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h. (H) Quantitation of the percentage of CD138 + cells within each division from (G). All data represent the combination of at least 2 independent experiments. Data points in (B) and (D) represent individual mice with horizontal bars depicting the data mean. Error bars in (F) and (H) depict mean ± SD. Statistics were calculated using a paired Student t- test.

Article Snippet: B cells were stimulated with either 20 μg/mL LPS (L2630; Sigma), 20 ng/mL IL-2 (575406; BioLegend), and 5 ng/mL IL-5 (581504; BioLegend) or 500 ng/mL CD40L (R&D Systems, 8230-CL), 10 ng/mL IL-4 (R&D Systems, 404-ML), and 10 ng/mL IL-5.

Techniques: Ex Vivo, Concentration Assay, Flow Cytometry, Quantitation Assay, Control

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme augments S phase cells at later divisions. (A) Naïve B cells were stained with CTY and stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24 h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (B) Naïve B cells were stained with CTY and stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (C) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (A). (D) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (B). Data represent the combination of at least 2 independent experiments. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Article Snippet: B cells were stimulated with either 20 μg/mL LPS (L2630; Sigma), 20 ng/mL IL-2 (575406; BioLegend), and 5 ng/mL IL-5 (581504; BioLegend) or 500 ng/mL CD40L (R&D Systems, 8230-CL), 10 ng/mL IL-4 (R&D Systems, 404-ML), and 10 ng/mL IL-5.

Techniques: Staining, Ex Vivo, Control, Flow Cytometry, Quantitation Assay, BrdU Incorporation Assay

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme represses the p21-Rb cell cycle regulatory axis. (A) Model depicting how p53, p21, and Rb function to regulate the G1 to S phase transition of the cell cycle under conditions of cell cycle arrest (left) or cell cycle progression (right). Naïve B cells were stimulated with CD40L, IL-4, and IL-5, and heme or vehicle control was added at 24 h. (B) Bar plot representing Trp53 transcript abundance at 54 h and 72 h measured by RT-qPCR. (C) Western blot for p53 protein and β-actin. Images for p53 protein were adjusted for brightness (+40%) and contrast (−40%). (D) Bar plot representing p53 expression relative to β-actin from (C). (E) Boxplot quantitating the ATAC-seq signal at p53 motifs in the indicated condition. (F) Genome plot of the Cdkn1a locus depicting the ATAC-seq signal and significant differential accessible region (box) in heme- vs vehicle-treated activated B cells (ActB) and plasma cells (PCs). The location of a previously identified p53 binding site is indicated in red. (G) Bar plot representing Cdkn1a transcript abundance at 54 h and 72 h measured by RT-qPCR. (H) Representative flow cytometry histogram showing intracellular staining for p21 protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (I) Bar plot quantitating MFI of p21 expression from (H). (J) Representative flow cytometry histogram showing intracellular staining for Rb protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (K) Bar plot quantitating MFI of Rb expression from (J). Data represent the combination of at least 2 independent experiments with the indicated P value calculated with paired Student t- tests. FMO, Fluorescence Minus One; MFI, median fluorescence intensity; RPPM, reads per peak per million.

Article Snippet: B cells were stimulated with either 20 μg/mL LPS (L2630; Sigma), 20 ng/mL IL-2 (575406; BioLegend), and 5 ng/mL IL-5 (581504; BioLegend) or 500 ng/mL CD40L (R&D Systems, 8230-CL), 10 ng/mL IL-4 (R&D Systems, 404-ML), and 10 ng/mL IL-5.

Techniques: Sublimation, Control, Quantitative RT-PCR, Western Blot, Expressing, Clinical Proteomics, Binding Assay, Flow Cytometry, Staining, Fluorescence