Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 3 HIF-1a and GPER are involved in the hypoxia-induced transcriptional activation of VEGF. (a) The mRNA expression of VEGF is up-regulated in CAFs and SkBr3 cells treated with 100 μM CoCl2, as evaluated by real-time PCR. Values are normalized to the 18S expression and shown as fold changes of mRNA expression induced by CoCl2 compared to cells treated with vehicle (-). Columns, mean of three independent experiments; bars, SD. (b) The VEGF promoter plasmid (pVEGF) is transactivated in CAFs and SkBr3 cells treated with 100 μM CoCl2 for 12 h (b) or exposed to low oxygen tension (2% O2) for 12 h (c). The transactivation of the VEGF promoter observed in SkBr3 cells treated for 12 h with 100 μM CoCl2 is abrogated by silencing HIF-1a (d) or GPER expression (e). The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle or cultured under normoxia were set as one-fold induction upon which the activities induced by CoCl2 treatment or hypoxia were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (○), (●) P < 0.05 for cells receiving vehicle (-) or cultured under normoxia vs cells treated with CoCl2 or cells cultured under hypoxia.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Transfection, Control, Cell Culture

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 4 Hypoxia induces VEGF protein expression in CAFs. Evaluation of VEGF expression by immunofluorescence assays. CAFs were treated for 12 h with vehicle (a2) or 100 μM CoCl2 (a3), or cultured under normoxia (b2) or low oxygen tension (2% O2 for 12 h) (b3), as indicated. VEGF accumulation is evidenced by the green signal. Nuclei were stained by DAPI (blue signal) (a1, b1). For descriptive purposes, each side panel shows the plot profiles obtained at the level of the yellow line of the corresponding inset using the program WCIF Image J for Windows. Note the higher values indicating zones of intense labeling. Images shown are representative of 20 random fields of three independent experiments.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Immunofluorescence, Cell Culture, Staining, Labeling

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 5 HIF-1a and GPER mediate the hypoxia-induced expression of VEGF in CAFs. Evaluation of VEGF expression by immunofluorescent microscopy in CAFs transfected for 24 h with control shRNA (a2, a3, b2, b3), shHIF-1a (a4, a5) or shGPER (b4, b5). Cells were treated with vehicle or 100 μM CoCl2 for 12 h, as indicated and VEGF accumulation is evidenced by the green signal. Nuclei were stained by DAPI (blue signal) (a1, b1). For descriptive purposes, each side panel shows the plot profiles obtained at the level of the yellow line of the corresponding inset using the program WCIF Image J for Windows. Note the higher values indicating zones of intense labeling. Images shown are representative of 20 random fields of three independent experiments.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Microscopy, Transfection, Control, shRNA, Staining, Labeling

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 6 GPER is involved in VEGF expression by hypoxia in SkBr3 breast cancer cells. (a) SkBr3 cells were treated for 12 h with vehicle (a2) or 100 μM CoCl2 (a3), as indicated. VEGF accumulation is evidenced by the green signal. (b) SkBr3 cells were transfected for 24 h with control shRNA (b2, b3) or shGPER (b4, b5) and treated with vehicle (b2, b4) or 100 μM CoCl2 (b3, b5) for 12 h, as indicated. VEGF accumulation was evidenced by the green signal. Nuclei were stained by DAPI (blue signal) (a1, b1). For descriptive purposes, each side panel shows the plot profiles obtained at the level of the yellow line of the corresponding inset using the program WCIF Image J for Windows. Note the higher values indicating zones of intense labeling. Images shown are representative of 20 random fields of three independent experiments.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Transfection, Control, shRNA, Staining, Labeling

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 7 GPER is involved in VEGF expression by hypoxia in HL-1 murine cardiomyocytes. (a) HL-1 cells were treated for 12 h with vehicle (a2) or 100 μM CoCl2 (a3), as indicated. VEGF accumulation is evidenced by the green signal. (b) HL-1 cells were transfected for 24 h with control shRNA (b2, b3) or shGPER (b4, b5) and treated with vehicle (b2, b4) or 100 μM CoCl2 (b3, b5) for 12 h, as indicated. VEGF accumulation is evidenced by the green signal. Nuclei were stained by DAPI (blue signal) (a1, b1). For descriptive purposes, each side panel shows the plot profiles obtained at the level of the yellow line of the corresponding inset using the program WCIF Image J for Windows. Note the higher values indicating zones of intense labeling. Images shown are representative of 20 random fields of three independent experiments.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Transfection, Control, shRNA, Staining, Labeling

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 8 HIF-1a and GPER are recruited to the VEGF promoter sequence. CAFs treated for 1 h with 100 μM CoCl2 were submitted to the chromatin immunoprecipitation procedure using anti-HIF-1a (a) or anti-GPER (b) antibodies. For knockdown experiments, CAFs were transfected with control shRNA and shGPER (c) or with control shRNA and shHIF-1a (d), treated for 1 h with 100 μM CoCl2 and then submitted to the chromatin immunoprecipitation procedure using anti-HIF-1a (c) or anti-GPER (d) antibodies. The amplified sequences were evaluated by real-time PCR. Each data point represents the mean ± SD of three independent experiments. (○) P < 0.05 for cells receiving vehicle vs CoCl2 treatment.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Sequencing, Chromatin Immunoprecipitation, Knockdown, Transfection, Control, shRNA, Amplification, Real-time Polymerase Chain Reaction

Journal: Breast cancer research : BCR

Article Title: HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs).

doi: 10.1186/bcr3458

Figure Lengend Snippet: Figure 9 Involvement of HIF-1a and GPER in hypoxia-induced tube formation. Tube formation was evaluated in HUVECs cultured for 2 h in medium collected from CAFs which were cultured under normoxia or hypoxia (2% O2 for 12 h). To this end, CAFs were transfected with control shRNA (a), shHIF-1a (b) or shGPER (c) and exposed to hypoxia, as indicated. Tube formation is rescued in HUVECs treated with 10 ng/mL VEGF and cultured in medium from CAFs transfected with shGPER and cultured under hypoxia (2% O2 for 12 h). Data are representative of three independent experiments performed in triplicate.

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology, DBA).

Techniques: Cell Culture, Transfection, Control, shRNA

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet: ( A ) Myo10 protein content increases during myoblast differentiation, as measured by immunoblotting (n = 3 individual experiments). ( B ) Fractionation of differentiation day 5 myoblast cultures into soluble and insoluble cellular fractions reveals that the slight majority of Myo10 content exists in the soluble fraction. ( C ) Immunofluorescence (IF) of differentiated myoblast insoluble fractions shows that Myo10 of the insoluble cellular fraction is associated with the actin cytoskeleton (as shown by phalloidin staining) and can be found at the tips of thin cellular projections. ( D ) Schematic of the consensus E-box-binding motifs (CANNTG) identified in the Myo10 promoter. ( E–F ) Activation of the Myo10 promoter reporter plasmid in differentiating myoblasts co-transfected with constitutively expressed GFP-CAAX and mApple (RFP) driven by the Myo10 promoter depicted in ( D ) (n = 4 individual experiments). ( G ) Expression of RFP in a differentiating myoblast following 1 day of differentiation. Efficient shRNA-mediated knockdown (KD) of myoblast Myo10 gene expression in ( H ) undifferentiated and ( I ) differentiated myoblasts, whereas muscle differentiation is not affected by Myo10 KD, as indicated by Myh2 expression, a gene encoding a mature myosin heavy chain (MHC) expressed by skeletal muscle (n = 3 individual experiments). ( J ) Representative images of MHC IF of control shRNA cells, Myo10 KD cells expressing a control RFP plasmid after 7 days of differentiation, and Myo10 KD cells expressing an RFP-Myo10 rescue plasmid. Data are presented as box-and-whisker plots depicting second and third quartiles with minimum and maximum values. Data of ( A ) were analyzed using one-way ANOVA followed by Tukey post hoc tests (α = 0.05; *p < 0.05 vs. day 1 values; # p < 0.05 vs. day 3 values; effect size is presented as eta-squared (η 2 )). Data of ( E ) and ( H–I ) were analyzed using two-tailed Welch’s t-tests with effect size presented as Cohen’s d ( d ). Scale bars represent ( G ) 10 or ( C, J ) 25 µm. Figure 2—figure supplement 1—source data 1. Source data file for . Figure 2—figure supplement 1—source data 2. Source data file for . Figure 2—figure supplement 1—source data 3. Source data file for . Figure 2—figure supplement 1—source data 4. Source data file for . Figure 2—figure supplement 1—source data 5. Source data file for . Figure 2—figure supplement 1—source data 6. Source data file for .

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Western Blot, Fractionation, Immunofluorescence, Staining, Binding Assay, Activation Assay, Plasmid Preparation, Transfection, Expressing, shRNA, Knockdown, Gene Expression, Control, Whisker Assay, Two Tailed Test

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet: Clonal lines of C2C12 cells expressing control or Myo10 -targeted short-hairpin RNA (shRNA) were validated for efficacy of Myo10 knockdown (KD) and myogenic differentiation potential. ( A ) Immunoblotting for Myo10 protein and the myogenic differentiation marker, myosin heavy chain (MHC; loading control visualized by Ponceau Red staining). KD of Myo10 myoblasts results in loss of filopodia during differentiation compared to control shRNA cells, as demonstrated by ( B ) immunofluorescence (day 3), ( C ) scanning electron microscopy (day 5), and ( D ) live-cell confocal microscopy (day 5), as well as loss of ( E ) cellular extension lengths (n = 31–152 cellular extensions). Myoblast differentiation assays (n = 3 individual experiments) reveal loss of multinucleated myotubes formation in Myo10 KD cells after 7 days of differentiation compared to control cells, quantified as ( F ) population distribution of myotube nuclear content. ( G–H ) Loss of fusion ability by Myo10 KD cells can be partially rescued by transfection of a full-length Myo10 construct with an N-terminal mApple fluorescent tag (RFP-Myo10; n = 3–6 individual experiments). Data analysis performed using ( E–F ) Welch’s two-tailed t-test (α = 0.05) with effect size displayed as Cohen’s d ( d ) or ( H ) one-way ANOVA followed by Tukey post hoc tests (α = 0.05; *p < 0.05 vs. control values; # p < 0.05 vs. RFP values; effect size is presented as eta-squared (η 2 )). Unless otherwise noted, scale bars represent 25 µm. Figure 3—source data 1. Source data file for . Figure 3—source data 2. Source data file for . Figure 3—source data 3. Source data file for . Figure 3—source data 4. Source data file for .

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Expressing, Control, shRNA, Knockdown, Western Blot, Marker, Staining, Immunofluorescence, Electron Microscopy, Confocal Microscopy, Transfection, Construct, Two Tailed Test

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet:

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Transfection, Construct, Membrane, Activation Assay, Control, shRNA, Sequencing

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris

doi: 10.1371/journal.pone.0151386

Figure Lengend Snippet: (A) Expression of TRPM3 protein in whole body of H . vulgaris . Western blot is representative of six independent experiments. (B) Immunofluorescence of Hydra whole mounts with the anti-TRPM3 antibody. Signals for TRPM3 are clearly extracellular and appear mainly in tentacles and foot. Specimens were examined in the confocal microscope. Scale bars, 100 μm.

Article Snippet: Membrane was incubated with rabbit polyclonal anti-TRPM3 antibody (O/N, 4°C, 1:100 dilution; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Western Blot, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris

doi: 10.1371/journal.pone.0151386

Figure Lengend Snippet: (A) A pretreatment for 10 min with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits HSP70 gene expression induced by heat shock (HS; 34°C). (B) TRPM3 agonist pregnenolone sulfate (PS; 10 μM) increase HSP70 gene expression, the peak being observed after 1.5 h. (C) MFA (20 μM) inhibits HSP70 gene expression induced by PS (10 μM). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (T = 0) ± SEM (n = 9). (A) * p≤0.01 vs CTRL and ^^ p≤0.05 vs HS; (B) * p≤0.01 vs 0; (C) * p≤0.01 vs CTRL and ^^ p≤0.05 vs PS.

Article Snippet: Membrane was incubated with rabbit polyclonal anti-TRPM3 antibody (O/N, 4°C, 1:100 dilution; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris

doi: 10.1371/journal.pone.0151386

Figure Lengend Snippet: Both HS (A) and TRPM3 agonist PS (C) induce an increase of SOD mRNA level, the maximal peak being observed after 24 h. A pretreatment with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits the effect relative to HS (B) or PS (D). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 h) ± SEM (n = 9). (A) * p≤0.01 vs 0; (B) ^ p≤0.05 vs CTRL and ^^ p≤0.05 vs HS; (C) ^ p≤0.05 vs 0; (D) ^ p≤0.05 vs CTRL and ^^ p≤0.05 vs PS.

Article Snippet: Membrane was incubated with rabbit polyclonal anti-TRPM3 antibody (O/N, 4°C, 1:100 dilution; Alomone Labs, Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris

doi: 10.1371/journal.pone.0151386

Figure Lengend Snippet: HS (A) and the TRPM3 agonist PS (C) time-dependently induce Nrf2 with a peak observed after 1.5 h. A pretreatment for 10 min with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits the effect relative to both HS (B) and PS (D). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 h) ± SEM (n = 9). (A) * p≤0.01 vs 0 and ^ p≤0.05 vs 0; (B) * p≤0.01 vs CTRL and ^^ p≤0.05 vs HS; (C) * p≤0.01 vs 0 and ^ p≤0.05 vs 0; (D) * p≤0.01 vs CTRL and ** p≤0.01 vs PS.

Article Snippet: Membrane was incubated with rabbit polyclonal anti-TRPM3 antibody (O/N, 4°C, 1:100 dilution; Alomone Labs, Jerusalem, Israel).

Techniques:

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Antibodies used for immunofluorescence staining of trigeminal ganglia and cornea sections.

Article Snippet: Mm00436469_m1 , Semaphorin 3A , Mus musculus.

Techniques: Immunofluorescence, Staining

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Gene expression assays used for qPCR analysis of axonal guidance proteins in the corneal epithelium.

Article Snippet: Mm00436469_m1 , Semaphorin 3A , Mus musculus.

Techniques: Gene Expression

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Basal and post-injury expression of axon guidance proteins in mouse cornea. Mice were subjected to corneal epithelium debridement, and the expression of these proteins was followed up in a time-dependent manner. All of these proteins are well-expressed in the uninjured cornea. After debridement, they quickly recovered, and their expression was visible from day 3 to day 14 post-injury. When compared to uninjured corneas, EphrinB1 was highly expressed at all post-injury days, while EphrinB2 and Sema3A where highly expressed from day 7 to day 14. The expression of Sema3F, Netrin-1 and Netrin-4 at day 14 was similar to the basal conditions. Quantification of immunostaining described in Methods is shown in the graphs. Representative images of the central debrided cornea, n = 3 mice per time point. Scale bar = 50 μm.

Article Snippet: Mm00436469_m1 , Semaphorin 3A , Mus musculus.

Techniques: Expressing, Immunostaining

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Variable gene expression recovery of axon guidance proteins in injured mouse cornea. Mouse corneal epithelium was harvested from uninjured mice corneas or animals that received cornea epithelium debridement. Gene expression was analyzed as described in Materials and Methods. We found that 3 days after injury, only the expression of EphrinB2 was significantly upregulated, while all other axon guidance ligands were downregulated. At later time points, Sema3A, EphrinB1, and EphrinB2 were upregulated as well, while Sema3F, Netrin-1 and Netrin-4 were downregulated. In general, all of these proteins trend to normalize their expression to the basal level, and only EphrinB1 was still showing high level of expression after 4 weeks post-injury. N = 5 mice per time point. * = p < 0.05.

Article Snippet: Mm00436469_m1 , Semaphorin 3A , Mus musculus.

Techniques: Gene Expression, Expressing

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Axon guidance proteins accelerated corneal epithelial wound closure. Human corneal limbal epithelial (HCLE) cells were grown to confluency on a 24-well plate and then a scratch was performed using a scratcher tip as described in Methods. Cells were then treated with recombinant Sema3A, EphrinB2, Netrin-4 or left untreated as control. Cells were immediately placed in a chamber incubator on a microscope and images were collected every 20 min for 15 h and analyzed using Fiji software. All the axon guidance proteins accelerated the wound closure when compared to control. (A) Representative images of scratch area. Sema3A (B) and EphrinB2 (C) induced faster healing with complete closure at 8 h, while Netrin-4 (D) did at 10 h, still faster than control. Each treatment was performed in triplicate and 3 images were obtained per treatment. Experiments were performed three times. Scale bar = 400 μm. * = p < 0.05; ** = p < 0.01. Values are mean ± S.E.M.

Article Snippet: Mm00436469_m1 , Semaphorin 3A , Mus musculus.

Techniques: Recombinant, Control, Microscopy, Software

Journal: Scientific Reports

Article Title: The TIPE Molecular Pilot That Directs Lymphocyte Migration in Health and Inflammation

doi: 10.1038/s41598-020-63629-w

Figure Lengend Snippet: Essential roles of TIPE proteins in chemokine-induced PtdIns(3,4,5)P 3 generation, and morphological and Rac1-GTP polarization. ( a ) PtdIns(3,4,5)P 3 levels detected by AKT-PH-GFP biosensor in live WT and DKO CD4 + T cells, in response to point source stimulation of CCL21, as visualized by super-resolution fluorescence video microscopy. Upper panels show a WT and a DKO T cells 60 sec after CCL21 stimulation, with arrows pointing to the source of CCL21. The lower panel shows the changes in PtdIns(3,4,5)P 3 levels relative to Time 0 when CCL21 was applied; values are mean ± s.e.m., with n = 20 cells for each group. ( b ) Morphological polarization of WT and DKO CD4 + T cells, in response to point source stimulation of CCL21, as visualized by phase contrast microscopy. Upper panels show a polarized WT and an unpolarized DKO T cells 10 min after CCL21 stimulation. The lower panel shows the percentages of polarized and unpolarized cells 10 min after CCL21 stimulation; n = 308 cells for WT and 304 cells for DKO group. ( c,d) Rac1-GTP polarization in WT and DKO CD4 + T cells, in response to point source stimulation of CCL21, as visualized by immunofluorescence microscopy. Panel-c shows WT and DKO T cells 60 sec after incubation with CCL21 or medium alone ( Control ). Panel-d shows the calculated polarization index of each group treated as in Panel-c; n = 16–24 cells for WT and 17–18 for DKO group. Values are mean ± s.e.m. The experiments were repeated independently at least three times ( a – d ) with similar results. *P < 0.05; **P < 0.01; *** *P < 0.0001 (Mann-Whitney U test ( a ) or Student’s t -test ( b , d )).

Article Snippet: Briefly, splenic CD4 + T cells were stimulated with anti-CD3 and anti-CD28 for 40 h, and then transfected with GFP-C1-AKT-PH vector (Addgene, Watertown, MA) using mouse T cell Nucleofector Kit and Amaxa Nucleofector II (program X-001, Lonza).

Techniques: Fluorescence, Microscopy, Immunofluorescence, Incubation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Amplified DNAs from junctional intra-σδ deletions as well as Sμ–σδ, Sμ–Sγ1 and Sμ–Sα1 recombinations from human tonsil B cells or human peripheral blood naïve IgM + IgD + B cells stimulated with CpG plus IL-2 and IL-21 and cultured for 120 h, OVA-immunized C57BL/6 mouse spleen B cells or C57BL/6 mouse naïve IgM + IgD + B cells stimulated with LPS plus IL-4 and cultured for 96 h were amplified and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in intra-σδ deletions, Sμ–σδ, Sμ–Sγ1, and Sμ–Sα1 junctional DNAs are shown by violin plots. Each dot represents a unique junctional sequence ( n = 45 per group). b Human and mouse Sμ and σδ regions consist of repetitive motifs, which are better-suited substrates for Rad52-mediated MMEJ than those in Sμ and Sγ1 or Sμ and Sα. As such, they can facilitate the formation of microhomologies. Repetitive sequence elements in mouse and human Sμ, σδ, Sγ1 and Sα that can potentially form microhomologies were identified by Pustell Matrix dot plot using MacVector software and are depicted by small dots. Intensity of dots depicts frequency and degree of complementarity of respective sequences. c Somatic point-mutations in Sμ and σδ regions abetting recombined Sμ−σδ DNA junctions in IgD class-switched human and mouse B cells in vivo and in vitro. Mutations were identified in a 48–506 nt stretch of Sμ or σδ regions in unique Sμ–σδ DNA recombination sequences. Each dot represents an individual sequence. Sequence data were pooled from three individuals in each group. Box and whiskers plots show median, quartiles, maximum and minimum of mutation frequencies in Sμ and σδ regions. In pie charts, the size of slices denotes the proportion of transcripts with the same number of mutations and the gray hue denotes the number of point mutations per transcript. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). ** p < 0.01, *** p < 0.001, ns: not significant (unpaired two-tailed t- test). Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Amplification, Cell Culture, Sequencing, Software, In Vivo, In Vitro, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Recombined Sμ–σδ, Sμ−Sγ1, Sμ−Sα, and Sμ−Sε DNAs in mouse Rad52 +/+ , Rad52 −/− and Aicda −/− naïve IgM + IgD + B cells stimulated with nil, LPS alone, LPS plus IL-4, LPS plus TGF-β and RA, CD154 alone, CD154 plus IL-4, or CD154 plus TGF-β and RA, as well as Sμ–Sγ3 in Rad52 +/+ , Rad52 −/− and Aicda −/− B cells stimulated with LPS only, were analyzed 96 h post-stimulation by specific nested PCR using forward Iμ and reverse Cδ, Sγ1, Sγ3, Sα, or Sε primers, respectively, followed by Southern-blotting using specific Sμ, σδ, Sγ1, Sγ3, Sα, or Sε probe, as indicated. Data are one representative of three independent experiments yielding comparable results. b IgD titers in culture (96 h) fluid of Rad52 +/+ , Rad52 −/− or Aicda −/− B cells stimulated with LPS plus IL-4, as measured by dot-blotting (two-fold serial diluted culture fluid) using a rat anti-mouse IgD mAb. Data are one representative of five independent experiments yielding comparable results. Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Nested PCR, Southern Blot

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: Rad52 +/+ and Rad52 −/− mice were immunized with OVA in alum i.p. a Recombined Sμ–σδ, Sμ−Sγ1, and Sμ−Sα DNAs in spleen, mesenteric lymph nodes (MLNs), and Peyer’s patches (PPs) B cells, as analyzed by nested PCR using forward Iμ and reverse Cδ, Sγ1, or Sα primers, respectively, followed by Southern-blotting using specific Sμ, σδ, Sγ1, or Sα probe, as indicated. Data are one representative of three independent experiments yielding comparable results. b Sμ–σδ, Sμ–Sγ1, and Sμ–Sα junctional DNAs were amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sμ–σδ, Sμ−Sγ1, and Sμ−Sα junctional DNAs are shown by violin plots. Each symbol represents a unique sequence ( n = 45 per group). c – f Titers of total IgD in serum, BALF, and feces, as analyzed by dot-blotting using rat anti-mouse IgD mAb—titers of total IgM, IgD, IgG1, and IgA as well as OVA-binding IgM, IgD, IgG1, and IgA as analyzed by specific ELISAs. Each dot represents datum from one individual mouse ( n = 5–8 per group, as indicated). Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant (unpaired two-tailed t -test). No adjustments were made for multiple comparisons. g Bacteria-bound IgD and IgA in feces as analyzed by flow cytometry. h IgM, IgD, and IgA positive cells in MLNs and lamina propria as visualized by fluorescence microscopy. Scale bar = 100 μm. Data in g , h are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Nested PCR, Southern Blot, Amplification, Sequencing, Binding Assay, Two Tailed Test, Bacteria, Flow Cytometry, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a C57BL/6 mouse naïve IgM + IgD + B cells were stimulated with LPS plus IL-4 and cultured for 0, 24, 48, 72 and 96 h. Rad52 , Ku70 , Ku86 , and Aicda transcripts were analyzed by real-time qRT-PCR, normalized to β-Actin expression, and depicted as relative to the expression in unstimulated B cells (set as 1.0). Data are mean ± SEM of three independent experiments. b Expression of Rad52, phosphorylated Rad52 (p-Rad52), AID, Ku70, Ku86, and β-Actin proteins in mouse B cells stimulated with LPS plus IL-4 (as in a ), as analyzed by specific immunoblotting. Data are one representative of three independent experiments yielding comparable results. c Human peripheral blood naive IgM + IgD + B cells were stimulated with CD154 plus IL-4 and IL-21 and cultured for 0, 24, 48, 72 and 96 h. RAD52 , KU70 , KU86 , and AICDA transcripts were analyzed by real-time qRT-PCR, normalized to β-ACTIN expression, and depicted as relative to the expression in unstimulated B cells (set as 1.0). Data are mean ± SEM of three independent experiments. d Recruitment of Rad52 to σδ region DNA, as analyzed by ChIP-qPCR assays in mouse Rad52 +/+ and Rad52 −/− B cells stimulated with LPS plus IL-4 and cultured for 72 h. Data are expressed as percent of pre-IP input for each sample (mean ± SEM of three independent experiments). *** p < 0.001 (unpaired two-tailed t -test). e , f C57BL/6 mouse naïve IgM + IgD + B cells were stimulated with nil, LPS alone, LPS plus IL-4, or LPS plus TGF-β and RA and cultured for 72 h. Recruitment of Rad52 ( e ) and Ku70/Ku86 ( f ) to Sμ, σδ, Sγ1, Sγ3, and Sα region DNA, as analyzed by ChIP-qPCR assays. Data are mean ± SEM of three or four independent experiments. Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, ChIP-qPCR, Two Tailed Test

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a C57BL/6 mouse naïve IgM + IgD + B cells were stimulated with nil, LPS plus IL-4 or LPS plus TGF-β and RA. Surface expression of IgM and IgD were analyzed 96 h post-stimulation by flow cytometry. Expression of V H DJ H −C δ m , V H DJ H −C δ s , V H DJ H −C μ m , and V H DJ H −C μ s transcripts were analyzed 72 h post-stimulation by semi-quantitative RT-PCR using serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. b IgD in supernatant from cultures (96 h) of C57BL/6 naïve IgM + IgD + B cell stimulated with nil, LPS plus IL-4, LPS plus TGF-β and RA, or CD154 plus IL-4, as analyzed by dot-blotting using rat anti-mouse IgD mAb. Data are representative of 5 independent experiments. c Expression of Zfp318 transcripts in mouse naïve B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by qRT-PCR and normalized to β-Actin expression and depicted relative to the average expression in unstimulated B cells (set as 1). Data are mean ± SEM of three independent experiments. ** p < 0.01, *** p < 0.001 (unpaired two-tailed t -test). d Expression of Zfp318 transcripts in unstimulated mouse naïve B cells (Nil) and mouse naïve B cells stimulated with LPS plus IL-4 for 72 h, as analyzed by mRNA-Seq. Data are mean ± SEM of four independent experiments. *** p < 0.001 (unpaired two-tailed t -test). e Zfp318 protein level in mouse naïve B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 96 h post-stimulation by intracellular staining with rabbit anti-Zfp318 Ab in flow cytometry. Bars in the right panel represent level of MFI (mean ± SEM) from three independent experiments. * p < 0.05, *** p < 0.001 (unpaired two-tailed t -test). f Human blood naïve IgM + IgD + B cells were stimulated with nil, CpG plus IL-2 and IL-21 or CpG plus IL-4 and IL-21; V H DJ H −C δ m , V H DJ H −C δ s, V H DJ H −C μ m , and V H DJ H −C μ s transcript levels were measured 72 h post-stimulation by semi-quantitative RT-PCR with serial two-fold dilution of cDNA templates—data are representative of three independent experiments (left panels). Expression of ZFP318 transcripts as analyzed 72 h post-stimulation by qRT-PCR and normalized to HPRT expression ( f middle panel)—data are mean ± SEM of three independent experiments. Secreted IgD in supernatants of the human B cell cultures, as analyzed 120 h post-stimulation by specific ELISA ( f right panel)—data are mean ± SEM of four independent experiments. ** p < 0.01, *** p < 0.001 (unpaired two-tailed t -test). g Expression of ZFP318 transcripts in human naïve CD27 − IgM + IgD + B cells and memory CD27 + IgM − IgD + B cells isolated from peripheral blood of healthy subjects, as analyzed by mRNA-Seq. Data are mean ± SEM of three independent experiments. h Expression of V H DJ H −C δ m, V H DJ H −C δ s, V H DJ H −C μ m , and V H DJ H −C μ s transcripts in human tonsil IgD + B cells, as analyzed by semi-quantitative RT-PCR involving serial two-fold dilution of cDNA templates (left panel)—data are representative of three independent experiments. Expression of ZFP318 protein in human tonsil IgM + IgD + B cells and IgM − IgD + B cells, as analyzed by intracellular staining with anti-Zfp318 Ab in flow cytometry (middle panel)—data are representative of four independent experiments (mean ± SEM, right panel). ** p < 0.01, ns: not significant (unpaired two-tailed t -test). i Surface expression of IgM and IgD in mouse naïve Rad52 +/+ , Rad52 −/− and Aicda −/− B cells stimulated with LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 96 h post-stimulation by flow cytometry. Data are representative of three independent experiments. j Expression of V H DJ H −C δ m and V H DJ H −C δ s transcripts in mouse naïve Rad52 +/+ and Rad52 −/− B cells stimulated with nil, LPS plus IL-4 or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by semi-quantitative RT-PCR using serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. k Rad52 or AID deficiency does not alter Zfp318 expression. Expression of Zfp318 transcripts in mouse naïve Rad52 +/+ , Rad52 −/− and Aicda −/− B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by qRT-PCR and normalized to β-Actin expression, as depicted relative to expression in unstimulated B cells (set as 1). Data are mean ± SEM of three independent experiments. No adjustments were made for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Human blood naïve IgM + IgD + B cells were transfected with specific RAD52 siRNA or scrambled (Scra) siRNA and stimulated by CpG plus IL-2 and IL-21. Recombined Sμ–σδ and Sμ–Sγ1 DNA in the transfected B cells 120 h after RAD52 siRNA transfection, as well as Sμ–σδ DNA in tonsil IgM – IgD + and blood naive IgM + IgD + B cells were analyzed by nested PCR using forward Iμ and reverse Cδ or Sγ1 primers followed by Southern-blotting using indicated specific probes. Data are from three independent experiments. b Expression of RAD52 and AICDA transcripts was analyzed 48 h after RAD52 siRNA or Scra siRNA transfection by qRT-PCR and normalized to HPRT expression. Data are mean ± SEM of three independent experiments. ** p < 0.01, ns: not significant (unpaired two-tailed t -test). c Expression of RAD52 and AID proteins were analyzed 72 h after RAD52 siRNA or Scra siRNA transfection by specific Western blotting. Data are representative of three independent experiments. d Expression of V H DJ H −C δ m and V H DJ H −C δ s transcripts as analyzed by semi-quantitative RT-PCR with of serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. e RAD52 is recruited to σδ region DNA in human B cells undergoing CSR to IgD. Recruitment of RAD52 to Sμ and σδ region DNA in human blood naive IgM + IgD + B cells stimulated for 120 h with CpG plus IL-2 and IL-21, as analyzed by specific ChIP-qPCR. Data are mean ± SEM of three or four independent experiments. * p < 0.05, ** p < 0.01 (unpaired two-tailed t -test). Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Transfection, Nested PCR, Southern Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, ChIP-qPCR

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Human blood naive IgM + IgD + B cells were stimulated with CpG plus IL-2 and IL-21, which induce IgD CSR, or CpG plus IL-4 and IL-21, which do not induce IgD CSR. Proportions of CD138 + IgM − IgD + plasmablasts/plasma cells among intracellular sIgM − IgD + B cells and BLIMP-1 expression in intracellular sIgM – IgD + cells, as analyzed 120 h post-stimulation by flow cytometry. Alexa Fluor 647-fluorescence minus one (FMO) controls are shown as reference. b Mouse Rad52 +/+ B cells and Rad52 −/− B cells stimulated with LPS plus IL-4, which induce IgD CSR. Proportions of sCD138 + plasmablasts/plasma cells among intracellular IgD + sIgM − cells and Blimp-1 expression in intracellular IgD + sIgM − cells, as analyzed 96 h post-stimulation by flow cytometry. FITC-FMO controls are shown as reference. Data in a and b are representative of three independent experiments. c Recombined Sμ–σδ and Sμ–Sα DNA in two IgD + myelomas and one IgA + myeloma, as analyzed by specific nested PCR followed by Southern-blotting using indicated probes.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Clinical Proteomics, Expressing, Flow Cytometry, Fluorescence, Nested PCR, Southern Blot

Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Serum total and double-strand DNA (dsDNA)-, RNA-, histone-, or RNP/Sm-binding IgD in healthy human subjects and systemic lupus erythematosus (SLE) patients, as analyzed by specific ELISAs. Each dot represents the datum from one individual human subject. Mean ± SEM of 6–10 healthy subjects or SLE patients are depicted. * p < 0.05 (unpaired two-tailed t -test). b Human and mouse antinuclear autoantibodies (ANAs), as visualized by indirect immunofluorescence microscopy on HEp-2 cells that were incubated with serum from a healthy human subject, an SLE patient, a C57BL/6 mouse, or a MRL/ Fas lp/lpr mouse, as revealed by FITC-labeled rat mAb to human or mouse IgD. Scale bar = 50 μm. c Total IgD in serum, feces and BALF as analyzed by dot-blotting, and concentrations of IgD autoantibodies to dsDNA or histone in serum of C57BL/6 and MRL/ Fas lpr/lpr mice, as analyzed by specific ELISAs. Each dot represents datum from one individual mouse. Data are mean ± SEM of 3–9 mice, as indicated. ** p < 0.01, *** p < 0.001 (unpaired two-tailed t -test). d IgD concentrations in serum, feces and BALF from C57BL/6 and MRL/ Fas lpr/lpr mice, as analyzed by dot-blots. Shown are dot-blots from one C57BL/6 and one MRL/ Fas lpr/lpr mouse, representative of 3–9 C57BL/6 and MRL/ Fas lpr/lpr mice. e Bacteria-bound IgD and IgA in feces from C57BL/6 and MRL/ Fas lpr/lpr mice, as analyzed by flow cytometry. f Expression of V H DJ H −C δ m, V H DJ H −C δ s, V H DJ H −C μ m , and V H DJ H −C μ s transcripts in bone marrow (BM), spleen and mesenteric lymph nodes (MLNs), as analyzed semi-quantitative RT-PCR by serial two-fold dilutions of cDNA templates. Shown are RT-PCR data from one C57BL/6 mouse and one MRL/ Fas lpr/lpr mouse, representative of three C57BL/6 and three MRL/ Fas lpr/lpr mice. g IgD + B cells in lamina propria, MLNs and Peyer’s patches (PPs) of C57BL/6 and MRL/ Fas lpr/lpr mice, as visualized by fluorescent microscopy. Scale bar = 100 μm. h Recombined junctional Sμ–σδ, Sμ–Sγ1, and Sμ–Sα DNAs in bone marrow, spleen, MLNs, and PPs B cells from C57BL/6 and MRL/ Fas lpr/lpr mice as analyzed by specific nested PCR using forward Iμ and reverse Cδ, Sγ1 or Sα primers, followed by Southern-blotting using indicated probes. Data are representative of three independent experiments. i Sμ–σδ, Sμ–Sγ1 and Sμ–Sα junctional DNAs in non-immunized MRL/ Fas lpr/lpr mice, as amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sμ–σδ, Sμ–Sγ1, and Sμ–Sα junctional DNAs are rendered by violin plots. Each dot represents a unique sequence ( n = 45 per group). j Somatic point-mutations in Sμ and σδ regions abetting recombined Sμ−σδ DNA junctions in IgD class-switched spleen B cells from three MRL/ Fas lpr/lpr mice. Mutations were identified in a 48–506 nt stretch of Sμ or σδ regions in unique Sμ–σδ DNA recombination sequences. Each dot represents an individual sequence. ns: not significant (unpaired two-tailed t -test). Box and whiskers plots show the median, quartiles, maximum, and minimum of mutation frequencies in Sμ and σδ regions. In pie charts, the size of slices denotes the proportion of sequences with the same number of mutations and the gray hue denotes the number of point-mutations per sequence. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). k Expression of phosphorylated Rad52 (p-Rad52), Rad52 and β-Actin proteins in peripheral blood B cells from healthy human subjects and SLE patients as well as B cells from C57BL/6 mice and MRL/ Fas lpr/lpr mice, as analyzed by specific Western blotting using rabbit anti-p-Rad52 Ab or anti-β-Actin mAb—p-Rd52 (Y104) Ab detected endogenous levels of Rad52 protein only when phosphorylated at tyrosine 104. l Expression of ZFP318 and AICDA transcripts in B cells from healthy human subjects and SLE patients (left panel), as well as Zfp318 and Aicda transcripts in MLNs from C57BL/6 and MRL/ Fas lpr/lpr mice (right panel), as analyzed by specific qRT-PCR. Data are mean ± SEM of three healthy human subjects, three SLE patients, three C57BL/6 mice, and three MRL/ Fas lpr/lpr mice. * p < 0.05 (unpaired two-tailed t -test). No adjustments were made for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Chromatin was fragmented by sonication (DNA fragments of about 200–1000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology, 5 μg ml −1 ), anti-Ku70/86 mAb (MA1-21818, Thermo Fisher Scientific, 5 μg ml −1 ), or control rabbit or mouse IgG mAb with irrelevant specificity at 4 °C overnight.

Techniques: Binding Assay, Two Tailed Test, Immunofluorescence, Microscopy, Incubation, Labeling, Bacteria, Flow Cytometry, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Nested PCR, Southern Blot, Amplification, Sequencing, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Impaired cholesterol transport from aged astrocytes to neurons can be rescued by cannabinoids

doi: 10.1101/2023.07.24.550299

Figure Lengend Snippet: A) Western blot analysis, along with quantification showing decreased NPC1 protein levels in aged primary hippocampal astrocytes compared to control cultures. B) RT-qPCR analysis showing decreased NPC1 mRNA levels aged rat primary hippocampal astrocytes. C) Western blot analysis, along with quantification showing decreased NPC1 protein levels in the hippocampus of old (20 months-old) mice compared to young (2 months-old) C57BL/6 mice (YOUNG = 1.0630 + 0.0364 SEM, OLD = 0.8919 + 0.0494 SEM, p = 0.0303). D) RT-qPCR analysis showing decreased NPC1 mRNA levels in the hippocampus of old C57BL/6 mice compared to young individuals (YOUNG = 1.0635 + 0.1290 SEM, OLD = 0.5290 + 0.1213 SEM, p = 0.0242). E) Quantification of miR-33 levels by RT-qPCR in control, aged, or H2O 2 -treated hippocampal astrocytes, indicating increased expression of miR-33 in these cells due to aging or oxidative stress. F) RT-qPCR analysis showing decreased NPC1 mRNA levels in hippocampal astrocytes treated with H 2 O 2 compared to untreated controls. G) RT-qPCR quantification of ABCA1 mRNA levels in control, aged, or H 2 O 2 -treated hippocampal astrocytes, demonstrating downregulation of ABCA1 in primary astrocytes due to aging or oxidative stress. H) Fluorescence microscopy images of rat primary hippocampal astrocytes treated with Probucol 10µM (ABCA1 inhibitor), U18666A 4µg/mL (NPC1 inhibitor), and the combination of both, labeled with Bodipy-cholesterol. I) Quantification of images including those shown in H, indicating that NPC1 or ABCA1 inhibition leads to Bodipy-cholesterol accumulation in astrocytes, with enhanced effects observed when both proteins are inhibited. J) Treatment of aged astrocytes with the ABCA1 agonist evodiamine leads to a significant decrease in the levels of accumulated Bodipy-cholesterol. Data are represented as Mean ± SEM. ****P-value < 0.0001, *P-value < 0.05.

Article Snippet: Membranes were blocked for 1 hour and incubated overnight at 4°C with the NPC1 primary antibody (rabbit polyclonal, Novus Biologicals, UK, NB 400-148).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Labeling, Inhibition

Journal: bioRxiv

Article Title: Impaired cholesterol transport from aged astrocytes to neurons can be rescued by cannabinoids

doi: 10.1101/2023.07.24.550299

Figure Lengend Snippet: A) Fluorescence microscopy images of aged rat hippocampal astrocytes labeled with Bodipy-cholesterol and treated with the cannabinoids Anandamide (AEA 2µM), 2-Arachidonoylglycerol (2AG 2µM) and cannabidiol (CBD 5µM). B) Quantification of Bodipy-cholesterol intensity from images as shown in A demonstrates that treatment of aged astrocytes with AEA, 2AG, or CBD significantly reduces the Bodipy-cholesterol accumulation characteristic of aged astrocytes. C) Immunofluorescence images showing the levels of Bodipy-cholesterol incorporated in 7DIV hippocampal neurons co- cultured with either control, aged, or aged astrocytes in the presence of AEA for 6 hours. Neuronal cells were labeled for β3-Tubulin. D) Quantification of Bodipy-cholesterol intensity from images including those in C demonstrates that the cholesterol uptake by neurons co-cultured with aged astrocytes is improved by addition of AEA. E) Fluorescence microscopy images showing the levels of Bodipy-cholesterol accumulated in control, U18666A+Probucol, and U18666A+Probucol+AEA treated primary astrocytes. F) Bodipy-cholesterol quantification of images as shown in E demonstrate that the cholesterol buildup triggered by the inhibition of NPC1 and ABCA1 in primary astrocytes is alleviated by either AEA, 2AG, or CBD addition even in the presence of the inhibitors (U18666A+Probucol). These data suggest that cannabinoids act through NPC1 and ABCA1-independent pathways. Data are represented as Mean ± SEM. ****P-value < 0.0001.

Article Snippet: Membranes were blocked for 1 hour and incubated overnight at 4°C with the NPC1 primary antibody (rabbit polyclonal, Novus Biologicals, UK, NB 400-148).

Techniques: Fluorescence, Microscopy, Labeling, Immunofluorescence, Cell Culture, Control, Inhibition

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Mouse (Ms; A, B, G, H) or human (Hm; C, D, G, H) blood polymorphonuclear neutrophils (PMN) or differentiated HL60 neutrophil-like cells dHL60 cells (E, F, G, I) were stained with far-red SiR-DNA to fluorescently label DNA, plated on coverslips, stimulated with 4 μM ionomycin, and imaged live by differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = +3 μm) at 1-2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted above images. Double-arrow-headed lines above images indicate cells that are in presence of ionomycin, boxes around images correspond to cellular event categories in the graphs, with the color of the box indicating the event with the same colored background in graphs. A, C, E: Time series of image overlays of DIC (greyscale) and fluorescence (red) of cell and DNA dynamics. B, D, F: Quantification of the percent of cells that exhibit microvesicle (MV) shedding, DNA de-condensation, nuclear rounding, release of nuclear DNA into the cytoplasm (DNA Release), loss of DIC contrast in the cell periphery suggesting plasma membrane permeabilization (PM Permeabilization), and extracellular DNA release (NETosis) after ionomycin stimulation determined from time-lapse DIC and confocal color-overlay movies. n = total number of cell observed, each point represents the percent of cells performing the event in one experiment, long bar = mean, short bars = S.D. G, H: Quantification of the timing of microvesicle shedding relative to ionomycin stimulation (G) or of cellular event initiation relative to microvesicle shedding (H) determined from time-lapse DIC and confocal color-overlay movies. n= total number of cells observed, points represent individual cells, mean (long bar) and S.D. (short bar) also shown below each plot. I: Time series of DIC (upper row-left) and fluorescence images of cell, plasma membrane (lower row-left) and histone H1 (right: upper row, green; lower row) dynamics in a living dHL60 cell stained with far-red SiR-DNA and co-expressing the farnesylation signal sequence CAAX fused to mApple (CAAX-mApple) as a plasma membrane marker and H1-mEmerald as a histone marker. Boxes around images indicate different cellular events (green: MV Shedding; yellow: DNA De-condensation; blue: DNA release to the cytosol, red: extracellular DNA release). A, C, E, I: bars = 10 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Staining, Confocal Microscopy, Fluorescence, Expressing, Sequencing, Marker

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Mouse (Ms; A, B) or human (Hm; C, H) blood polymorphonuclear neutrophils (PMN) or dHL60 cells (D, E, F, G, I, J) were plated on coverslips, stimulated with 4 μM ionomycin and fixed (panels A, C) at different time points after stimulation (noted on images) or imaged live (panels F,G,H, I) by differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = + 3 μm) at 1-2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted above images. Double-arrow-headed lines above images indicate cells that are in presence of ionomycin, blue arrowheads mark rupture points in the lamina or NE. D, G-I: Green boxes around images indicate MV shedding. A: Immunofluorescence of lamin B1/B2 (upper row and bottom row, green) and DAPI (at 10 min) or SiR-DNA (DMSO, 5, 60 and 240 min) staining of DNA (bottom row, red) in fixed Ms PMN. B: Quantification from immunostaining experiments of the percent (numbers in bar) of Ms PMN exhibiting discontinuities in the lamin B1/B2 meshwork at different time points after stimulation, 0 represent cells treated with DMSO only. n = total number of observed cells. C: Immunofluorescence of lamin B1 (middle row and bottom row, green) and DAPI staining of DNA (bottom row, red) in Hm PMN fixed after 5 min of DMSO or 5, 10, 60 and 240 min of ionomyocin treatment. Z-stacks of 0.2 μm steps were acquired on a spinning-disk confocal microscope. Top row: Phase contrast images at the bottom plane (Z = 0 μm); Middle and bottom rows: Confocal images at the bottom plane (Z = 0 μm) and 1 or 3 μm above in the cell center (Z = + 1 μm, Z = + 3 μm). D: Time-series of DIC (top row) and confocal (lower rows) images of live dHL60 cells expressing lamin A-mEmerald (middle row and bottom row, green) and stained with far-red SiR-DNA to fluorescently label DNA (SiR-DNA, bottom row, red). E: Quantification of the percent of cells exhibiting rupture of the nuclear lamina at one or more than one points determined from movies of dHL60 cells expressing lamin A-mEmerald. n = total number of cell observed, each point represents the percent of cells in one experiment, long bar = mean, short bars = S.D. C: F, G, H: Time-series of DIC (upper rows) and confocal (lower rows) images of live dHL60 cells (F, G) expressing the KDEL sequence combined with the ER-retention signal sequence from calreticulin fused to mEmerald (ER-mEmerald) (F, bottom row; G, middle and bottom row, green) or live Hm PMN (H) stained with ER-Tracker Red (H, middle rows and bottom row, green) to label the ER and nuclear envelope (NE) and stained with SiR-DNA (G, H, bottom rows red). I: Time-series of DIC (upper rows) and confocal (lower rows) images of live dHL60 cells co-expressing the inner nuclear envelope protein, Lap2β-mEmerald (second row and bottom row, green) and lamin B1-mApple (third row and bottom row, blue) and stained with SiR-DNA (bottom row, red). In (G, H,I) the blue arrowheads highlight site of lamina and NE rupture. J: Quantification of the timing of the initiation of ER vesiculation or rupture of the nuclear lamina (Lamin B1 or Lamin A Rupture), the inner NE (Lap2β Rupture) or outer NE (ER (ONM) Rupture) relative to microvesicle shedding determined from time-lapse DIC and confocal color-overlay movies of dHL60 cells. n = total number of cell observed, points represent individual cells, mean (long bar) and S.D. (short bar) shown below each plot. **** and * for P value respectively < 0.0001, and <0.1; ns for non-significant. Statistical test: Mann-Whitney Test. A, C, D, F-I: bars = 5 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Immunostaining, Microscopy, Expressing, Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Human blood polymorphonuclear neutrophils (Hm PMN; A, C, E) or differentiated HL60 neutrophil-like cells (dHL60; B, D, F, G-I) were plated on coverslips, stimulated with 4 μM ionomycin and fixed at different time points after stimulation (noted above images, E) or imaged live by differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = +3 μm) at 1-2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted above images. Double-arrow-headed lines above images indicate cells that are in presence of ionomycin. A-F: Time-series of DIC (A-D and F) or phase contrast (E) (top row) and confocal (lower rows) images of live (A, C) or fixed (E) Hm PMN or live dHL60 cells (B, D, F). A, B: Cells were stained with far red SiR-actin (A) or expressing F-Tractin-mApple (B) to fluorescently label actin filaments. C, D: Cells were stained with far red SiR-tubulin (C) or expressing the ensconsin microtubule binding domain fused to eGFP (D) to fluorescently label microtubules. E, F: Cells were fixed and vimentin was immunolocalized (E) or were expressing vimentin-mEmerald (F) to fluorescently label vimentin intermediate filaments. A-D, F: Green boxes around images indicate MV Shedding. G: Quantification of the timing of the initiation of actin and vimentin disassembly (Dis) and end of microtubule disassembly relative to microvesicle shedding determined from time-lapse DIC and confocal color-overlay movies of dHL60 cells. n = total number of cell observed, points represent individual cells, mean (long bar) and S.D. (short bar) shown below each plot. H, I: Quantification of the percent of cells that exhibit actin depolymerization (Actin Disassembly), microvesicle (MV) shedding, DNA de-condensation, nuclear rounding, release of nuclear DNA into the cytoplasm (DNA Release), loss of DIC contrast in the cell periphery suggesting plasma membrane permeabilization (PM Permeabilization), and extracellular DNA release (NETosis) after ionomycin stimulation determined from time-lapse DIC and confocal color-overlay movies of cells treated with vehicle (Control) or jasplakinolide (1 μM, H) or taxol (10 μM, I) to prevent actin and microtubule disassembly, respectively. n = total number of cell observed, points represent the percent of cells in one experiment, long bar = mean, short bars = S.D. Statistical test: Fisher’s Exact Test. A-F: bars = 5 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Confocal Microscopy, Staining, Expressing, Binding Assay

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Differentiated HL60 neutrophil-like cells (dHL60; A-H) or human blood polymorphonuclear neutrophils (Hm PMN; I, J) were plated on coverslips, stimulated with 4 μM ionomycin imaged live by differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = + 3 μm) at 1-2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted above images. Double-arrow-headed lines above images indicate cells that are in presence of ionomycin, boxes around images indicate different cellular events (green: MV Shedding; yellow: DNA De-condensation; blue: DNA release to the cytosol, red: extracellular DNA release). A: Time series of DIC (upper row-Left) and fluorescence images of cell, plasma membrane (lower row-left and right, magenta), and histone (upper row-right and bottom row-right, yellow) dynamics in a dHL60 cell co-expressing the farnesylation signal sequence CAAX fused to mApple (CAAX-mApple) as a plasma membrane marker and the histones marker H2B-mEmerald. B, D, F, I: Time series of DIC (upper row) and fluorescence (lower rows) images of cell and soluble fluorescent markers (calcein (622 Da, B, I, greyscale), Alexa fluor 647 (D, F, red) or Alexa fluor 594 (I, red) 10 kDa dextran or Oregon Green 488 70 kDa (F, green)) added to the imaging media, in living dHL60 (B, D, F) or Hm PMN (I) cells. C, E, G, J: Normalized intracellular fluorescence (to average extracellular level) of calcein (C, J, green), 10 kDa (E, G, J, red) or 70 kDa (G, green) dextran over time (in min) measured from time-lapse movies. Time points of DNA de-condensation determined from the DIC channel of the movies is noted on the graphs. H: Quantification of the onset of calcein, 10 or 70 kDa fluorescent dextran cellular entry relative to microvesicle shedding determined from time-lapse DIC and confocal color-overlay movies of dHL60 cells. n = total number of cell observed, points represent individual cells, mean (long bar) and S.D. (short bar) shown below each plot. A, B, D, F, I: bars = 10 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Confocal Microscopy, Fluorescence, Expressing, Sequencing, Marker, Imaging

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Differentiated HL60 neutrophil-like cells (dHL60) or human blood polymorphonuclear neutrophils (Hm PMN) were plated on coverslips and fixed (A, B) or stimulated with 4 μM ionomycin and imaged by phase-contrast or differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = + 3 μm) at 2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted above images. A, B: Phase-contrast (Phase C), DAPI staining of DNA (blue) and immunolocalization of PAD4 by two different antibodies (α-Pad4; 1 and 2, red and green) in fixed dHL60 (A) and Hm PMN (B). C: Quantification from immunostaining experiments of the mean nuclear to cytosolic ratio of PAD4 intensity using two different antibodies. n = total number of cells observed, points represent individual cells, mean (long bar) and S.D. (short bar) shown below each plot. D: Time series of DIC (top row) and fluorescence images (bottom rows) of a dHL60 cell and fluorescent PAD4 (middle row and bottom rows, green), a soluble nucleoplasmic marker (third row, red) and DNA (bottom row, blue) dynamics observed in a dHL60 cell stained with far red SiR-DNA to fluorescently label DNA and co-expressing PAD4-mEmerald and the nuclear localization signal sequence fused to mCherry (NLS-mCherry). Double-arrow-headed lines above images indicate cells that are in presence of ionomycin, boxes around images indicate different cellular events (green: MV Shedding; blue: DNA release to the cytosol). E-F: Normalized (to the maximum intensity in the compartment) mean nuclear (E) and cytosolic (F) over time (in min) of PAD4-mEmerald and NLS-mCherry measured from time-lapse movie of the cell in (D). Time point of loss of DIC contrast in the cell periphery suggesting plasma membrane permeabilization (PM Permeabilization) determined from the DIC channel of the movie is noted on the graphs. A, B, D: bars = 5 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Confocal Microscopy, Staining, Immunostaining, Fluorescence, Marker, Expressing, Sequencing

Journal: bioRxiv

Article Title: NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

doi: 10.1101/663427

Figure Lengend Snippet: Differentiated HL60 neutrophil-like cells (dHL60 WT) or dHL60 with the PAD4 gene modified by CRSPR-Cas9 gene editing (PAD4 CR dHL60), were stained with far red SiR-DNA to fluorescently label DNA, plated on coverslips, stimulated with 4 μM ionomycin, and imaged live by differential interference contrast (DIC) and spinning-disk confocal microscopy at the coverslip-cell interface (Z = 0 μm) and 3 μm above in the cell center (Z = + 3 μm) at 2 min intervals for 4 hours, with time in min relative to plasma membrane microvesicle shedding noted on images. Double-arrow-headed lines above images indicate cells that are in presence of ionomycin, boxes around images indicate different cellular events (green: MV Shedding; yellow: DNA de-condensation; grey: nuclear rounding; blue: DNA release to the cytosol, red: extracellular DNA release). A-F: Time series of image overlays of DIC (greyscale) and fluorescence (red) of cell and DNA dynamics. C-F: PAD4 CR dHL60 cells were expressing mEmerald-PAD4 (C; bottom row, green), mEmerald-Only (mEmerald-C1; D; bottom row, green), enzymatically dead PAD4 mutant (mEmerald-PAD4-C645A; E; bottom row, green) or the nuclear localization mutated PAD4 (mEmerald-PAD4-K59A/K60A/K61A; F; bottom row, green). G: Quantification of the percent of cells (Left) that exhibit microvesicle (MV) shedding, DNA de-condensation, nuclear rounding, loss of DIC contrast in the cell periphery suggesting plasma membrane permeabilization (PM Permeabilization), release of nuclear DNA into the cytoplasm (DNA Release), and extracellular DNA release (NETosis) or timing relative to microvesicle shedding (right) of DNA de-condensation, DNA release and NETosis after ionomycin stimulation as determined from time-lapse DIC and confocal color-overlay movies. n = total number of cell observed, each point represents the percent of cells in one experiment (left), or individual cells (right). long bar = mean, short bars = S.D. shown below each plot (right). H-K: Time-series of DIC (upper rows) and confocal (lower rows) images of live WT or PAD4 CR dHL60 cells expressing the inner nuclear envelope protein, Lap2β-mEmerald (H, I; bottom rows, green) or lamin B1-mEmerald (J, K; bottom rows, green) and stained with SiR-DNA (bottom rows red). L: Quantification of the timing relative to microvesicle shedding of the onset of inner nuclear envelope rupture (Lap2β Rupture) or lamina rupture (LaminB1 Rupture). n = total number of cells observed, points represent individual cells, mean (long bar) and S.D. (short bar) shown below each plot. G, L: **** and ** for Pvalue respectively < 0.0001, and <0.01; ns for non-significant. Statistical test (G-left and L): Mann-Whitney Test. A-F, H-K: bars = 10 μm.

Article Snippet: The so obtained PAD4 insert and the mEmerald-C1 vector were cut with restriction enzymes (KpnI, BamHI, New England Biolabs) then ligated together with T4 DNA ligase (ThermoFisher, EL001) after dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP; ThermoFisher, 18009019) of the linearized plasmid and gel purification (QIAquick Gel Extraction Kit; Quiagen, 28706) of both insert and vector.

Techniques: Modification, Staining, Confocal Microscopy, Fluorescence, Expressing, Mutagenesis, MANN-WHITNEY

Journal: PLoS Biology

Article Title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

doi: 10.1371/journal.pbio.1001819

Figure Lengend Snippet: (A) Cellular extracts from HCT116 cells stably expressing vector or FLAG-JMJD6 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry. (B) HCT116 cell lysates were immunoprecipitated with antibodies against JMJD6 followed by immunoblotting with antibodies against p53 (FL-393), or they were immunoprecipitated with antibodies against p53 (FL-393) followed by immunoblotting with antibodies against JMJD6. (C) Immunofluorescence-stained endogenous JMJD6 (red) and p53 (green) were visualized by confocal microscopy. DAPI staining was included to visualize the cell nucleus (blue). Scale bar, 25 µm. (D) Mapping the domain of p53 that is required for its interaction with JMJD6. GST pull-down experiments were performed with GST-fused JMJD6 and in vitro transcribed/translated Myc-tagged full-length p53 or deletions of p53.

Article Snippet: The sources of antibodies against the following proteins were as follows: FLAG (M2) and β-actin from Sigma, MDMX and acetyl-p53(p53 K382ac ) from Abcam, PUMA from Cell Signaling, p53 monoclonal antibody agarose conjugate p53 (FL-393) and JMJD6 from Santa Cruz, and p53 (DO-1) and p21 from MBL.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Immunofluorescence, Confocal Microscopy, In Vitro

Journal: PLoS Biology

Article Title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

doi: 10.1371/journal.pbio.1001819

Figure Lengend Snippet: (A) Recombinant p53 was incubated with or without recombinant JMJD6 in the presence or absence of α-ketoglutarate (2-OG) and Fe(II). The mixture was then separated on SDS-PAGE, and the band corresponding to the molecular weight of p53 was excised and digested with trypsin and analyzed by LC-MS/MS. Inserts show the doubly charged peptide precursor ions that were fragmented. The relevant ion fragments are labeled, and the corresponding peptide positions are illustrated. K, lysine; K(OH), hydroxylated lysine; the expected increase in mass by hydroxylation modification is 16 Dalton. M, methionine; m, randomly oxidized methionine, which results in a +16 Dalton shift in mass. (I) Experimental group with 2-OG, Fe(II), and JMJD6; (II) negative control group without JMJD6; (III) negative control group without Fe(II); (IV) negative control group without 2-OG. (B) Wild-type JMJD6 hydroxylates p53 381–393 at K382 of p53 in the presence of 2-OG and Fe(II) in vitro . The peptides corresponding to amino acids 381–393 of p53 (wild-type p53, p53K382R, or p53K382A) were incubated with or without recombinant JMJD6 or JMJD6(H187A/D189A) in the presence or absence of 2-OG and Fe(II) for 2 h at 37°C. The mixture was then analyzed by MALDI/TOF. (C) Hydroxylation of p53 at K382 in vivo . Lysates from HCT116 cells were immunoprecipitated with anti-p53 monoclonal antibody-conjugated agarose. Bound proteins were eluted with p53 peptide, separated on SDS-PAGE, and analyzed by LC-MS/MS. Inserts show the doubly charged peptide precursor ions that were fragmented. The relevant ion fragments are labeled and the corresponding peptide positions are illustrated. Analysis by LC-MS/MS revealed the presence of modified p53 382–393 peptide (M+2H) 2+ containing hydroxylation of K382. (D) Extracted ion chromatogram (XIC) of nonmodified (upper panel) and hydroxylated p53K382 (lower panel) extracted from vector (black) or JMJD6 (red) transfected HCT116 cells.

Article Snippet: The sources of antibodies against the following proteins were as follows: FLAG (M2) and β-actin from Sigma, MDMX and acetyl-p53(p53 K382ac ) from Abcam, PUMA from Cell Signaling, p53 monoclonal antibody agarose conjugate p53 (FL-393) and JMJD6 from Santa Cruz, and p53 (DO-1) and p21 from MBL.

Techniques: Recombinant, Incubation, SDS Page, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Labeling, Modification, Negative Control, In Vitro, In Vivo, Immunoprecipitation, Plasmid Preparation, Transfection

Journal: PLoS Biology

Article Title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

doi: 10.1371/journal.pbio.1001819

Figure Lengend Snippet: (A) HCT116 p53 +/+ or HCT116 p53 −/− cells were transfected with control siRNA or JMJD6 siRNAs. The mRNA levels of p21 and PUMA were detected by real-time RT PCR and the levels of the indicated proteins were detected by Western blotting. (B) HCT116 p53 +/+ or HCT116 p53 −/− cells were transfected with control siRNA or JMJD6 siRNAs together with p21 promoter-driven luciferase construct. Cells were then harvested and luciferase activity was measured and normalized to that of renilla. Each bar represents the mean ± S.D. for triplicate experiments. (C) HCT116 p53 −/− cells were transfected with control siRNA or JMJD6 siRNAs and were synchronized by double thymidine block and released into the cell cycle before cell cycle analysis by flow cytometry. Experiments were repeated three times and the data from a representative experiment are shown. (D) HCT116 p53 −/− cells were transfected with control siRNA or JMJD6 siRNAs, and annexin V/PI staining and flow cytometry were performed to assess cell apoptosis. (E) HCT116 p53 +/+ or HCT116 p53 −/− cells were infected with lentivirus carrying a control siRNA or JMJD6 siRNAs, and were subcutaneously injected into the right anterior armpit of BALB/c nude mice. Tumors were measured weekly with Vernier calipers, and volume was calculated using the formula π/6×length×width 2 . Each point represents the mean ± S.D. for different animal measurements ( n = 6). The levels of the indicated proteins extracted from xenograft tumor were detected by Western blotting. p values were determined by Student's t test. * p <0.01. (F) Immunohistochemical staining was performed in xenograft tumor sections using antibody against Ki-67. Scale bar, 36 µm. Proliferation was assessed by counting the number of Ki-67 positively stained nuclei and total number of cancer cells at 400× magnification in five representative regions of the tumor.

Article Snippet: The sources of antibodies against the following proteins were as follows: FLAG (M2) and β-actin from Sigma, MDMX and acetyl-p53(p53 K382ac ) from Abcam, PUMA from Cell Signaling, p53 monoclonal antibody agarose conjugate p53 (FL-393) and JMJD6 from Santa Cruz, and p53 (DO-1) and p21 from MBL.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Luciferase, Construct, Activity Assay, Blocking Assay, Cell Cycle Assay, Flow Cytometry, Staining, Infection, Injection, Immunohistochemical staining

Journal: PLoS Biology

Article Title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

doi: 10.1371/journal.pbio.1001819

Figure Lengend Snippet: (A) Knockdown of JMJD6 promotes acetylation of p53 K382 . Lysates from HCT116 cells treated with control siRNA or JMJD6 siRNA-1 were immunoprecipitated with antibodies against p53 followed by immunoblotting with antibodies against acetyl-K382 of p53 (p53 K382ac ). (B) HCT116 cells were transfected with CBP and different amounts of FLAG-JMJD6 or FLAG-JMJD6(H187A/D189A) (mut-JMJD6) expression constructs together with p21 promoter-driven luciferase reporter. Cells were then harvested and luciferase activity was measured and normalized to that of renilla. Each bar represents the mean ± S.D. for triplicate experiments. (C) The peptide p53 381–393 without (upper) or with (lower) K382 acetylation was incubated with recombinant JMJD6 in the presence of 2-OG and Fe(II). The mixture was then analyzed by MALDI/TOF. (D) qChIP assay was done to measure p53 K382ac bound to its response element and to a control site in p21 promoter in HCT116 cells treated with control siRNA or JMJD6 siRNA-1 and challenged with or without VP-16. (E) Lysates from HCT116 cells treated with control siRNA or JMJD6 siRNA-1 were immunoprecipitated with antibodies against p53 followed by immunoblotting with antibodies against MDMX or MDM2. (F) JMJD6 did not affect p53 ubiquitination in vivo . HCT116 p53 −/− cells were co-transfected with FLAG-JMJD6, HA-ubiquitin, wild-type p53, or p53 mutant (p53K382R). Forty-eight hours after transfection, cells were treated with MG132 for 6 h before cellular extracts were prepared for co-immunoprecipitation assays with anti-p53 followed by immunoblotting with anti-HA. (G) JMJD6 could not hydroxylate K382-methylated p53 381–393 peptides. The peptide p53 381–393 with or without K382 monomethylation was incubated with recombinant JMJD6 in the presence of α-ketoglutarate and Fe(II). The mixture was then analyzed by MALDI/TOF. (I) JMJD6 hydroxylates K382 of p53 381–393 peptides; (II) JMJD6 could not hydroxylate K382-methylated p53 381–393 peptides.

Article Snippet: The sources of antibodies against the following proteins were as follows: FLAG (M2) and β-actin from Sigma, MDMX and acetyl-p53(p53 K382ac ) from Abcam, PUMA from Cell Signaling, p53 monoclonal antibody agarose conjugate p53 (FL-393) and JMJD6 from Santa Cruz, and p53 (DO-1) and p21 from MBL.

Techniques: Knockdown, Control, Immunoprecipitation, Western Blot, Transfection, Expressing, Construct, Luciferase, Activity Assay, Incubation, Recombinant, Ubiquitin Proteomics, In Vivo, Mutagenesis, Methylation