Journal: The Journal of Immunology Author Choice

Article Title: SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors

doi: 10.4049/jimmunol.1801618

Figure Lengend Snippet: SSB2 function is dispensable for murine B cells. SSB2-deficient mice were generated from targeted ES cells, and B cell compartments and ex vivo–cultured cells from these mice were analyzed for alterations in development, proliferation, and DDR activation. ( A ) Scheme visualizing the targeted disruption of the murine Ssb2 gene using the GT technique. Integration of the GT cassette into exon 2 disrupts the Ssb2 open reading frame (see also ). ( B ) Western blot of whole-cell lysates from mature B cells, confirming SSB2 protein loss in Ssb2 −/− mice. ( C ) Image of Ssb2 −/− and wild-type mice. ( D ) Bar diagram showing absolute numbers of CD19 + B cells from BM and spleens of Ssb2 −/− (black) and wild-type (gray) mice determined by flow cytometry ( n = 3). ( E and F ) Bar diagrams showing absolute numbers of BM (E) and splenic (F) B cell compartments in Ssb2 −/− and wild-type mice ( n = 3) defined by flow cytometric analysis of BM and spleens. Error bars indicate SEM. BM B cell compartments are those defined by Hardy and Hayakawa and characterized by surface expression of B220, CD19, CD24, and CD43. Splenic B cell fractions were defined by CD19, CD21, and CD23. Gating strategies and backgating to define B cell subsets are shown in . ( G ) Representative images of CFSE-stained, ex vivo–cultured, and IL-7–stimulated BM B cells (upper panel) and of LPS/IL-4–stimulated splenic B cells (lower panel) from Ssb2 −/− and wild-type mice ( n = 3). The days of analysis post-CFSE labeling are indicated. ( H ) Analysis of NHEJ-dependent Ig CSR toward IgG1 in LPS/IL-4–stimulated splenic B cells from Ssb2 −/− and wild-type mice determined by flow cytometry. Representative dot plots (upper panel) and a bar diagram (lower panel, n = 4) are shown. ( I ) Analysis of DNA damage–induced G 2 /M cell cycle arrest in irradiated splenic B cells from Ssb2 −/− mice (1 Gy, 1 h recovery) by flow cytometric detection of intracellular serine 10–phosphorylated Histone 3. ( J ) Western blot analysis of DNA damage–induced p-P53 Ser15 , p-CHK1 Ser317 , and p-H2AX Ser139 using whole-cell lysates from LPS/IL-4–stimulated splenic B cells (IR: 5 Gy, 1 h recovery). For (I) and (J), cells from Ssb2 +/− mice were used as Ctrl, and representative images of three independent experiments are shown.

Article Snippet: For detection of specific proteins by Western blot, the following Abs were used: anti-phospho-histone H2A.X (serine 139) (γH2AX) clone JBW301 (Millipore), anti-phospho-P53 (serine 15) (Cell Signaling), anti-phospho-CHK1 (serine 317) (R&D), anti-β-ACTIN (clone W16197A; BioLegend), anti-α-TUBULIN (Abcam), anti-LAMIN B1 (Abcam), anti-hSSB1/OBFC2B (SSB1) (Bethyl Laboratories), anti-OBFC2A (SSB2) (Proteintech Group), anti-phospho-KAP1 (serine 824) (Bethyl Laboratories), anti-FLAG-HRP (Sigma-Aldrich), anti-RPA1 (Bethyl Laboratories), anti-RPA2 (clone NA19L; Calbiochem), anti-RPA3 (Abcam), anti-Vinculin (Cell Signaling), and anti-BCL2 (Cell Signaling).

Techniques: Generated, Ex Vivo, Cell Culture, Activation Assay, Disruption, Western Blot, Flow Cytometry, Expressing, Staining, Labeling, Irradiation

Journal: The Journal of Immunology Author Choice

Article Title: SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors

doi: 10.4049/jimmunol.1801618

Figure Lengend Snippet: Combined loss of SSB1 and SSB2 impairs embryogenesis. The effect of combined loss of SSB1/2 protein on embryonic development was monitored by analysis of embryos at midgestation. Ssb1 +/− Ssb2 +/− mice were interbred and analyzed for their offspring at gestation days E10.5–E13.5. A total of 60 embryos were analyzed. ( A ) Summary of the embryo genotypes observed. ( B ) Representative images of Ssb1 +/− Ssb2 +/− , Ssb1 −/− Ssb2 +/+ , and Ssb1 −/− Ssb2 +/− embryos. Insets indicate normal hind limbs in Ssb1 +/− Ssb2 +/− embryos and early-stage hind limb defects in Ssb1 −/− Ssb2 +/+ embryos, as described .

Article Snippet: For detection of specific proteins by Western blot, the following Abs were used: anti-phospho-histone H2A.X (serine 139) (γH2AX) clone JBW301 (Millipore), anti-phospho-P53 (serine 15) (Cell Signaling), anti-phospho-CHK1 (serine 317) (R&D), anti-β-ACTIN (clone W16197A; BioLegend), anti-α-TUBULIN (Abcam), anti-LAMIN B1 (Abcam), anti-hSSB1/OBFC2B (SSB1) (Bethyl Laboratories), anti-OBFC2A (SSB2) (Proteintech Group), anti-phospho-KAP1 (serine 824) (Bethyl Laboratories), anti-FLAG-HRP (Sigma-Aldrich), anti-RPA1 (Bethyl Laboratories), anti-RPA2 (clone NA19L; Calbiochem), anti-RPA3 (Abcam), anti-Vinculin (Cell Signaling), and anti-BCL2 (Cell Signaling).

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors

doi: 10.4049/jimmunol.1801618

Figure Lengend Snippet: Combined Ssb1 / 2 deficiency impairs early B cell development and is differentially tolerated by immature and mature B cells. ( A – C ) Single cells from BM of Cd19Cre / + cDKO mice ( Ssb1flox/flox ; Ssb2 −/− ; Cd19Cre / + or Ssb1flox / − ; Ssb2 −/− ; Cd19Cre / + ) and Ctrl mice ( Ssb1 +/+ ; Ssb2 −/− ; Cd19Cre / + ) were analyzed by flow cytometry. (A) Representative flow cytometry plots (left) and a bar diagram indicating total BM B cell numbers (right) in Cd19Cre / + cDKO and Ctrl mice are shown (Ctrl, n = 4 and cDKO, n = 6 mice from >3 analyses). The inset Western blot indicates reduced SSB1 protein levels in BM B cells from Cd19Cre / + cDKO mice. (B) Absolute numbers of BM B cell compartments in Cd19Cre / + cDKO and Ctrl mice are shown (cDKO, n = 6 and Ctrl, n = 4; gating strategies and backgating to define B cell subsets are shown in ). (C) Representative dot blot images (left) and bar diagrams (right) showing absolute numbers of BM B cells with or without expression of sIg (IgM + and IgM/D + ) (DKO, n = 6 and Ctrl, n = 4). ( D – F ) Analysis of IL-7–cultured BM B cells from Cre-ERT2 cDKO ( Ssb1flox/flox ; Ssb2 −/− ; Cre-ERT2 ) and Ctrl mice ( Ssb1 +/+ ; Ssb2 −/− ; Cre-ERT2 ). 4-hydroxytamoxifen (OHT) was used to induce CRE recombinase activation and DKO induction. (D) Representative Western blots are shown, confirming DKO induction. Wild-type B cells were blotted as positive Ctrl for the anti-SSB2 Ab. (E and F) Analysis of proliferation and viability, respectively. (E, left) Representative histograms of CFSE-stained BM B cells are shown with time points of analysis indicated. Bar diagrams indicate proliferation (E) and viability (F) at day 6 after OHT addition ( n = 14 mice per group analyzed in >3 experiments). ( G – I ) Analysis of LPS/IL-4–cultured splenic B cells from Cre-ERT2 cDKO and Ctrl mice as in (D)–(F). (G) Representative Western blots are shown confirming OHT-induced SSB1/2 loss in splenic B cells. Diagrams in (H) and (I) indicate proliferation and viability as in (E) and (F) at day 3 after OHT addition and represent n = 6 (Ctrl) and n = 8 mice (cDKO) analyzed in >3 experiments. (H, left) Representative histograms of CFSE-stained splenic B cells are shown, with time points of analysis indicated. Error bars in (A)–(F) indicate SEM; statistical analysis was performed using two-tailed t test. ( J , left) Representative Western blot images are shown, confirming the inability of the SSB1-W55A and SSB1-Y74A mutants to bind ssDNA, as described . B cell precursors (middle) and mature-B cells (right) from BM and spleens of Ssb2 −/− mice were transduced with SSB1-WT, SSB1-W55A, and SSB1-Y74A encoding retrovirus (RV), and percentages of live transduced cells were monitored by flow cytometry for RV-encoded GFP expression at indicated time points ( n = 4 experiments, error bars indicate SD, statistical analysis was performed using the two-tailed t test).

Article Snippet: For detection of specific proteins by Western blot, the following Abs were used: anti-phospho-histone H2A.X (serine 139) (γH2AX) clone JBW301 (Millipore), anti-phospho-P53 (serine 15) (Cell Signaling), anti-phospho-CHK1 (serine 317) (R&D), anti-β-ACTIN (clone W16197A; BioLegend), anti-α-TUBULIN (Abcam), anti-LAMIN B1 (Abcam), anti-hSSB1/OBFC2B (SSB1) (Bethyl Laboratories), anti-OBFC2A (SSB2) (Proteintech Group), anti-phospho-KAP1 (serine 824) (Bethyl Laboratories), anti-FLAG-HRP (Sigma-Aldrich), anti-RPA1 (Bethyl Laboratories), anti-RPA2 (clone NA19L; Calbiochem), anti-RPA3 (Abcam), anti-Vinculin (Cell Signaling), and anti-BCL2 (Cell Signaling).

Techniques: Flow Cytometry, Western Blot, Dot Blot, Expressing, Cell Culture, Activation Assay, Staining, Two Tailed Test, Transduction

Journal: The Journal of Immunology Author Choice

Article Title: SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors

doi: 10.4049/jimmunol.1801618

Figure Lengend Snippet: Genetic loss of both SSB proteins can be rescued by overexpression of either SSB1 or SSB2 and suppressed by expression of BCL2 and alters cell cycle progression. Primary B cell precursors from Cre-ERT2 cDKO mice ( Ssb1flox/flox ; Ssb2 −/− ; Cre-ERT2 ) and Ctrl mice ( Ssb1 +/+ ; Ssb2 −/− ; Cre-ERT2 ) were immortalized by ectopic expression of c-MYC and used for the analysis of SSB1/2 loss on survival or cycle progression. ( A ) Immortalized cDKO B cell precursors were transduced with SSB1-WT, SSB1-W55A, or SSB1-Y74A encoding retrovirus (RV), and percentages of live transduced cells were monitored by flow cytometry for RV-encoded DSRED expression. ( B ) Immortalized cDKO B cell precursors were stably transduced with wild-type SSB1 or SSB2 encoding RV, or EV as Ctrl, and viability was measured at indicated time points after OHT-induced DKO induction. The inset shows a representative Western blot confirming DKO induction and forced expression of SSB1-WT and SSB2-WT in OHT-treated DKO cells. ( C ) Analysis of OHT- and mock-treated (MeOH) cDKO and Ctrl cells by flow cytometry using Annexin V and 7-AAD dyes. Representative dot blots (left) and a bar diagram (right) summarizing the results of n = 3 experiments are shown using one Ctrl and two independent cDKO cell lines each experiment. ( D ) Viability of DKO cells ectopically expressing BCL2 measured at indicated time points after the start of OHT treatment. EV-transduced DKO cells were used as Ctrl. ( E ) Cell cycle distribution of BrdU pulse-labeled and 7-AAD–labeled OHT- and mock-treated cDKO, Ctrl, and BCL2-expressing cells determined by flow cytometry. Left, Representative dot plots of BrdU/7-AAD staining. Right, Bar diagram indicating fractions of cells in individual cell cycle stages for the conditions indicated. Data represent n = 4 experiments using one Ctrl and two independent cDKO cell lines for each experiment. For experiments shown in (C) and (E), cells were treated for 2–3 d with OHT, reincubated in normal medium, and analyzed on day 5 post-OHT addition. For experiments shown in (B) and (D), analysis was performed at time points as indicated. Error bars indicate SD, and statistical analysis was done using two-tailed t test.

Article Snippet: For detection of specific proteins by Western blot, the following Abs were used: anti-phospho-histone H2A.X (serine 139) (γH2AX) clone JBW301 (Millipore), anti-phospho-P53 (serine 15) (Cell Signaling), anti-phospho-CHK1 (serine 317) (R&D), anti-β-ACTIN (clone W16197A; BioLegend), anti-α-TUBULIN (Abcam), anti-LAMIN B1 (Abcam), anti-hSSB1/OBFC2B (SSB1) (Bethyl Laboratories), anti-OBFC2A (SSB2) (Proteintech Group), anti-phospho-KAP1 (serine 824) (Bethyl Laboratories), anti-FLAG-HRP (Sigma-Aldrich), anti-RPA1 (Bethyl Laboratories), anti-RPA2 (clone NA19L; Calbiochem), anti-RPA3 (Abcam), anti-Vinculin (Cell Signaling), and anti-BCL2 (Cell Signaling).

Techniques: Over Expression, Expressing, Transduction, Flow Cytometry, Stable Transfection, Western Blot, Labeling, Staining, Two Tailed Test

Journal: The Journal of Immunology Author Choice

Article Title: SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors

doi: 10.4049/jimmunol.1801618

Figure Lengend Snippet: SSB1/2 loss in B cell precursors increases nuclear ssDNA exposure, disrupts genome fragile sites, and causes DNA damage if apoptosis is suppressed. Immortalized cDKO ( Ssb1flox/flox ; Ssb2 −/− ; Cre-ERT2 ) and Ctrl ( Ssb1 +/+ ; Ssb2 −/− ; Cre-ERT2 ) B cell precursors were analyzed for nuclear ssDNA exposure, fragile site disruption, and ssDNA breaks upon DKO induction. ( A ) BCL2-expressing cDKO and Ctrl cells were OHT- or mock-treated for 2 d, reincubated in normal medium, and BrdU labeled. Images of extracted nuclei stained under nondenaturing conditions using anti-BrdU Abs were taken 4 d after the start of experiment. A bar diagram representing n = 5 experiments (>100 nuclei per experiment) (left) and a representative image of a cell with >10 native BrdU foci are shown (right). ( B ) DNA-FISH analysis of the B cell–specific fragile site Pax5 and a nonfragile locus ( Foxp2 ) using locus-specific break-apart probes. For each locus, red- (excitation 547 nm, emission 565 nm) and green-labeled (excitation 495 nm, emission 517 nm) probes were used that cover individual parts of the locus and yield in one yellow signal if the locus is intact. Analysis was performed on interphases of OHT-treated cDKO and Ctrl cells, treated as described in . Left, Representative images of nuclei with intact and disrupted loci are shown. Disrupted loci are indicated by white arrows. Right, Fractions of interphases with nonintact loci were counted for the conditions indicated ( n = 3 experiments with at least 150 interphases per condition and experiment; error bars represent SD, unpaired student’s t test was employed to test significance). ( C ) DNA-FISH analysis of telomere integrity using cyanine 3 (Cy3)-labeled telomere-specific probes (red) and DAPI (blue). Cells were treated as above, fixed, and metaphase spreads were prepared. Representative images of reported telomere fragility phenotypes, such as smear, doublet, or triplet signals (indicated by white arrows) are shown (left) and summarized for n = 4 experiments for the conditions indicated (right). Quantification of % fragility per metaphase is presented as box plot of interquartile range; bars are defined as range from 0 to 99%, with the highest values of the data set depicted as dots. At least 40 metaphases per condition and experiment were analyzed. For analysis of SSB1-W55A–expressing cells, 33 metaphases were analyzed, obtained from three experiments. ( D ) Quantification of DNA breakage in OHT-treated cDKO and Ctrl cells by comet assay ( n = 3). DNA breakage per cell is given as the DNA comet tail moment. One hundred cells per condition were analyzed in each experiment. As positive Ctrl, cells were treated with CPT, HU, or APH. ( E ) Western blot analysis of MYC-immortalized cDKO and Ctrl B cell precursors for serine 15–phosphorylated P53, BCL2, SSB1, and lamin B1 as loading Ctrl. Cells used were stably transduced with BCL2 expression vector or EV and OHT-treated as in .

Article Snippet: For detection of specific proteins by Western blot, the following Abs were used: anti-phospho-histone H2A.X (serine 139) (γH2AX) clone JBW301 (Millipore), anti-phospho-P53 (serine 15) (Cell Signaling), anti-phospho-CHK1 (serine 317) (R&D), anti-β-ACTIN (clone W16197A; BioLegend), anti-α-TUBULIN (Abcam), anti-LAMIN B1 (Abcam), anti-hSSB1/OBFC2B (SSB1) (Bethyl Laboratories), anti-OBFC2A (SSB2) (Proteintech Group), anti-phospho-KAP1 (serine 824) (Bethyl Laboratories), anti-FLAG-HRP (Sigma-Aldrich), anti-RPA1 (Bethyl Laboratories), anti-RPA2 (clone NA19L; Calbiochem), anti-RPA3 (Abcam), anti-Vinculin (Cell Signaling), and anti-BCL2 (Cell Signaling).

Techniques: Disruption, Expressing, Labeling, Staining, Single Cell Gel Electrophoresis, Western Blot, Stable Transfection, Transduction, Plasmid Preparation