Journal: PLoS Biology

Article Title: The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility

doi: 10.1371/journal.pbio.3001584

Figure Lengend Snippet: (A) Testes ( n = 15) from <8-hour-old males of w Mel+, w Mel−, and transgenic cifAB lines were dissected and immunostained to visualize and quantify spermatid bundles with histone retention (purple) during late canoe stage of spermiogenesis. DAPI stain (blue) was used to label spermatid nuclei. Total spermatid bundles with DAPI signals and those with retained histones were manually counted and graphed. Compared to the negative control w Mel−, w Mel+ Wolbachia and dually expressed cifAB transgenic lines show abnormal histone retention in the late canoe stage. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant ( p < 0.05) differences as determined by pairwise comparisons based on Kolmogorov–Smirnov test. (B) Mature sperms isolated from seminal vesicles ( n = 15) of <8-hour-old males reared at 21°C were stained with fluorescent CMA3 (green) for detection of protamine deficiency in each individual sperm nucleus. Individual sperm head intensity was quantified in ImageJ (see ) and graphed. w Mel+ and transgenic cifAB lines show enhanced protamine deficiency levels compared to w Mel− control. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant ( p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p -values are reported in . The experiments were performed in 2 independent biological replicates and samples were blind-coded for the first run. Raw data underlying this figure can be found in file. CMA3, chromomycin A3.

Article Snippet: Significant differences ( p < 0.05) were determined by pairwise Mann Whitney U tests or by a Kruskal–Wallis test and Dunn multiple test correction in GraphPad Prism 7.

Techniques: Transgenic Assay, Staining, Negative Control, Standard Deviation, Isolation, Control

Journal: PLoS Biology

Article Title: The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility

doi: 10.1371/journal.pbio.3001584

Figure Lengend Snippet: (A) Sperms from the Wolbachia -infected (ΔProt+) and Wolbachia -uninfected (ΔProt−) protamine mutant (w[1118]; ΔMst35B[floxed], Sco/CyO) males exhibit significantly increased CMA3 fluorescence indicative of protamine deficiency compared to both wild-type w Mel+ and w Mel−. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant ( p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p -values are reported in . (B) CI hatch rate analyses of male siblings used in CMA3 assays (panel A) validate that ΔProt+ males with increased sperm protamine deficiency cause stronger (rescuable) CI levels than w Mel+. ΔProt− males do not cause CI. Asterisks indicate statistically significant ( p < 0.05) differences as determined by pairwise Mann–Whitney tests. All of the p -values related to CI assay are reported in . Raw data underlying this figure can be found in file. CI, cytoplasmic incompatibility; CMA3, chromomycin A3.

Article Snippet: Significant differences ( p < 0.05) were determined by pairwise Mann Whitney U tests or by a Kruskal–Wallis test and Dunn multiple test correction in GraphPad Prism 7.

Techniques: Infection, Mutagenesis, Fluorescence, Standard Deviation, MANN-WHITNEY

Journal: PLoS Biology

Article Title: The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility

doi: 10.1371/journal.pbio.3001584

Figure Lengend Snippet: (A) Schematic representation of CifA annotation shows the annotated bNLS with engineered amino acid substitutions and deletions. (B, C) Hatch rate assays assessed both CI (B) and rescue (C) in flies expressing wild-type, transgenic, and mutant cifA . Each dot represents the percent of embryos that hatched from a single male and female pair. Sample size is listed in parentheses. Horizontal bars represent the median. Letters to the right indicate significant differences determined by a Kruskal–Wallis test and Dunn multiple comparison tests. All the p -values are reported in . (D) Antibody labeling (green) and DAPI staining of onion stage spermatids in the testes of the bNLS mutant line ( cifA ΔbNLS ) reveals that the deletion ablates CifA’s localization to the nucleus, and CifA thus remains in the surrounding cytoplasm. The imaging experiment was conducted in parallel to nos;cifAB line shown in . (E) Mature sperms isolated from seminal vesicles ( n = 15) of <8-hour-old males of transgenic cifA Δbnls B line shows reduced fluorescence indicative of less Protamine deficiency compared to cifAB . To control for any background confounding effects of nos-Gal4VP16 driver line, w Mel− fathers were prior crossed to nos- mothers to generate males with nos;wMel− genotype. CMA3 fluorescence levels of sperms isolated from nos;wMel− males were similar to wMel− wild-type lines used in previous assays in this study. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant ( p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p -values are reported in , and raw data underlying this figure can be found in file. bNLS, bipartite nuclear localization signal; CI, cytoplasmic incompatibility.

Article Snippet: Significant differences ( p < 0.05) were determined by pairwise Mann Whitney U tests or by a Kruskal–Wallis test and Dunn multiple test correction in GraphPad Prism 7.

Techniques: Expressing, Transgenic Assay, Mutagenesis, Comparison, Antibody Labeling, Staining, Imaging, Isolation, Fluorescence, Control, Standard Deviation

Journal: PLoS Biology

Article Title: The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility

doi: 10.1371/journal.pbio.3001584

Figure Lengend Snippet: (A) Schematic representation of Drosophila melanogaster female reproductive system. Mature oocytes leave the OV and reach the UT, where they can be fertilized prior to being laid. Sperms from males are stored in specialized organs—SP and SR shown in the box, which open into the UT for fertilization to occur. Schematic is created with BioRender.com . (B) Transgenic cifAB -expressing and w Mel− males were crossed to w Mel− females. Four hours postfertilization, sperms isolated from females were decondensed and immunostained for localizing CifA (green) and CifB (red). DAPI stain (blue) was used to label nuclei. CifA is absent in sperm heads (empty arrowheads) and puctae are seen along the sperm tails (arrows). CifB is present in apical acrosomal tip of all of the sperm heads (solid arrowheads), with more distant signal in the more decondensed sperm nuclei. No Cifs are present in the sperms transferred from w Mel− negative control males. (C) Individual sperm intensity quantification shows that protamine deficiency of sperms from w Mel+ and transgenic cifAB males persists after transfer in the females compared to w Mel− males. Sperm protamine deficiency from transgenic cifAB males also persists in the reproductive tract of w Mel+ females. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant ( p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p -values are reported in , and raw data underlying this panel can be found in file. (D) Representative images of CMA3-stained mature sperms (arrows) transferred from w Mel−, w Mel+ and transgenic cifAB males in w Mel− and w Mel+ female reproductive systems are shown. CMA3, chromomycin A3; OV, ovary; SP, spermathecae; SR, seminal receptacle; UT, uterus.

Article Snippet: Significant differences ( p < 0.05) were determined by pairwise Mann Whitney U tests or by a Kruskal–Wallis test and Dunn multiple test correction in GraphPad Prism 7.

Techniques: Transgenic Assay, Expressing, Isolation, Staining, Negative Control, Standard Deviation

Journal: Retrovirology

Article Title: A detailed analysis of F-MuLV- and SFFV-infected cells in Friend virus-infected mice reveals the contribution of both F-MuLV- and SFFV-infected cells to the interleukin-10 host response

doi: 10.1186/s12977-022-00613-4

Figure Lengend Snippet: F-MuLV- and SFFV-infected cells in infected BALB/c mice. Highly susceptible BALB/c mice were infected with 500 SFFU of FV-Katushka-mTagBFP, and infected erythroblasts (Ery), B cells, Gr1 − (Gr1 − My) and Gr1 + myeloid cells (Gr1 + My), dendritic cells (DC), CD4 + T cells and CD8 + T cells in the bone marrow ( A ), spleen ( B ) and lymph nodes ( C ) were analyzed by flow cytometry on days 4, 7, 10, 14 and 21. The frequencies of F-MuLV-Katushka single-infected, SFFV-mTagBFP single-infected and F-MuLV-Katushka, SFFV-mTagBFP double-infected as well as uninfected cells are shown as indicated. Each dot indicates an individual mouse, bars indicate median values, the dotted line indicates the detection limit. Data from 6 (days 4, 7, 10, 21) or 9 (day 14) mice per group were obtained in 1–2 independent experiments per time point. Asterisk indicates statistically significant differences between the indicated types of single or double-infected cells ( p < 0.05, multiple t-test with Bonferroni correction for multiple comparisons). Number sign indicates dominant subsets of single or double-infected cells in the respective organ, i.e. infected cell types that were significantly higher than at least three other cell types of the same single or double-infected state ( p < 0.05, Kruskal–Wallis ANOVA on Ranks, Dunn’s post test). Dagger indicates statistically significant differences between the respective cell subsets in the bone marrow and spleen, double dagger indicates statistically significant differences between the respective cell subsets in the bone marrow and lymph nodes, inverted cross symbol indicates statistically significant differences between the respective cell subsets in the spleen and lymph nodes ( p < 0.05, multiple t-test with Bonferroni correction for multiple comparisons)

Article Snippet: Analysis of data for statistically significant differences was performed either using an unpaired t test for the comparison of parametric data from two groups or using a nonparametric one-way analysis of variance on ranks with Dunn’s multiple-comparison procedure in GraphPad Prism 8 software for the comparison of more than two groups.

Techniques: Infection, Flow Cytometry

Journal: Retrovirology

Article Title: A detailed analysis of F-MuLV- and SFFV-infected cells in Friend virus-infected mice reveals the contribution of both F-MuLV- and SFFV-infected cells to the interleukin-10 host response

doi: 10.1186/s12977-022-00613-4

Figure Lengend Snippet: F-MuLV- and SFFV-infected cells in IL-10 reporter mice. Susceptible IL-10-eGFP reporter mice of BALB/c background were infected with 500 SFFU of FV-Katushka-mTagBFP and infected erythroblasts (Ery), B cells, Gr1 − (Gr1 − My) and Gr1 + myeloid cells (Gr1 + My), dendritic cells (DC), CD4 + T cells and CD8 + T cells in the bone marrow ( A ), spleen ( B ) and lymph nodes ( C ) were analyzed by flow cytometry on days 2, 4, 7, 10, 14 and 28. The frequencies of F-MuLV-Katushka single-infected, SFFV-mTagBFP single-infected and F-MuLV-Katushka, SFFV-mTagBFP double-infected as well as uninfected cells are shown as indicated. Each dot indicates an individual mouse, bars indicate median values, the dotted line indicates the detection limit. Data from 7 (day 14), 8 (day 7) or 10 (day 2, 4, 10, 28) mice per group were obtained in 1–2 independent experiments per time point. Asterisk indicates statistically significant differences between the indicated types of single or double-infected cells ( p < 0.05, multiple t-test with Bonferroni correction for multiple comparisons). Number sign indicates dominant subsets of single or double-infected cells in the respective organ, i.e. infected cell types that were significantly higher than at least three other cell types of the same single or double-infected state ( p < 0.05, Kruskal–Wallis ANOVA on Ranks, Dunn’s post test). Dagger indicates statistically significant differences between the respective cell subsets in the bone marrow and spleen, double dagger indicates statistically significant differences between the respective cell subsets in the bone marrow and lymph nodes, inverted cross symbol indicates statistically significant differences between the respective cell subsets in the spleen and lymph nodes ( p < 0.05, multiple t-test with Bonferroni correction for multiple comparisons). Up-pointing triangle indicates statistically significant differences compared with the respective cell subsets in BALB/c mice ( p < 0.05, multiple t-test with Bonferroni correction for multiple comparisons)

Article Snippet: Analysis of data for statistically significant differences was performed either using an unpaired t test for the comparison of parametric data from two groups or using a nonparametric one-way analysis of variance on ranks with Dunn’s multiple-comparison procedure in GraphPad Prism 8 software for the comparison of more than two groups.

Techniques: Infection, Flow Cytometry

Journal: Retrovirology

Article Title: A detailed analysis of F-MuLV- and SFFV-infected cells in Friend virus-infected mice reveals the contribution of both F-MuLV- and SFFV-infected cells to the interleukin-10 host response

doi: 10.1186/s12977-022-00613-4

Figure Lengend Snippet: eGFP expression in infected and uninfected cells in IL-10-eGFP reporter mice. The infected and uninfected cells shown in Fig. were analyzed for the expression of eGFP as an indicator of IL-10 expression. The frequency of eGFP + cells in the respective cell population is shown. Each dot indicates an individual mouse, bars indicate median values. Tilde indicates infected cell subsets for which no percentage of eGFP + cells could be calculated for one or more mice as no infected cells were detected. Data from 7 (day 14), 8 (day 7) or 10 (day 2, 4, 10 28) mice per group were obtained in 1–2 independent experiments per time point. Asterisk indicates statistically significant differences between the indicated infected types of single or double-infected cells compared to uninfected cells ( p < 0.05, Kruskal–Wallis One Way ANOVA on Ranks, Dunn’s post test)

Article Snippet: Analysis of data for statistically significant differences was performed either using an unpaired t test for the comparison of parametric data from two groups or using a nonparametric one-way analysis of variance on ranks with Dunn’s multiple-comparison procedure in GraphPad Prism 8 software for the comparison of more than two groups.

Techniques: Expressing, Infection

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: Tumor growth assessment: data for each time-point in mm 3 are mean ± SEM. ( A ) M-234p, N = 6/group; Day 21: Control (600.96 ± 93) vs Cy+Los (20.96 ± 9.01) ( P < 0.05); ( B ) M-406, N = 6–7/group; Day 17: Control (1397.00 ± 328.32) vs Cy (372.00 ± 55.25) ( P < 0.01), vs Los (451.07 ± 143.94) ( P < 0.05), vs Cy+Los (123.43 ± 45.71) ( P < 0.001). Kruskal-Wallis multiple comparison test and Dunn’s post-test. Overall survival (Kaplan-Meier), Median Survival (MS): ( C ) M-234p, N = 5–6/group; Control (MS: 34 days); Cy (MS: 47 days); Los (MS: 32 days); Cy+Los (MS: undefined, Day 32: 60% [3/5] complete tumor regressions). Cy+Los vs Control, vs Los, vs Cy ( P < 0.01); ( D ) M-406, N = 6–7/group); Control (MS: 24 days); Cy (MS: 36.5 days); Los (MS: 33 days); Cy+Los (MS: 47 days). Control vs Cy ( P < 0.01), vs Los ( P < 0.01), vs Cy+Los ( P < 0.001); Cy+Los vs Cy ( P < 0.05), vs Los ( P < 0,001). Log-rank Test.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: Proliferation: Ki67 + cells/field (median, range). ( A ) M-234p Control vs Cy+Los ( P < 0.01); ( B ) M-406 Control vs Cy+Los ( P < 0.05; ( C ) M-234p and ( D ) M-406, representative images of Control and Cy+Los treated tumors, 1000× magnification. Apoptosis: TUNEL + cells/field (median, range). ( E ) M-234p Control vs Cy+Los ( P < 0.05): ( F ) M-406 N. S; Kruskal-Wallis multiple comparison test and Dunn’s post-test; ( G ) M-234p and ( H ) M-406 representative images of Control and Cy+Los treated tumors, 1000× magnification.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, TUNEL Assay, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: Hematoxylin and eosin (H&E) representative tumor sections from M-234p and M-406, 400×. In both models the behavior was similar. Control group: ( A ) M-234p and ( C ) M-406: capillaries with small endothelial cells with barely stained nuclei and intercellular gaps (yellow arrow), lack of pericytes or cells with structure and staining compatible with pericytes. Cy+Los group: ( B ) M-234p and ( D ) M-406: intra- and peritumoral capillaries with structure and morphology similar to normal tissues. Endothelial cells with defined nuclei provide a continuous uninterrupted lining (yellow arrow), and well defined basal membrane covered with pericytes (red arrow). M-406 magnified section (1000×): vessel with normal vascular morphology. HIF1α expression: HIF1α + cells/field (median, range). ( E ) Control vs Cy ( P < 0.05), ( F ) Control vs Cy ( P < 0.05), vs Cy+Los ( P < 0.05), ( G ) and H ), representative images of Control and Cy+Los treated tumors, 100× magnification. Kruskal-Wallis multiple comparison test and Dunn’s post-test.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, Staining, Membrane, Expressing, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: M-234p: ( A ) CD4 cells, N. S. ( B ) CD8 cells, N. S. ( C ) Treg cells, N. S. ( D ) Th17 cells, N. S. M-406: ( E ) CD4 cells, N. S. ( F ) CD8 cells, N. S. ( G ) Treg cells, ( P = 0.064). ( H ) Th17 cells: Control vs Cy+Los, ( P = 0.0580). Kruskal-Wallis multiple comparison test and Dunn’s post-test.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: Lymphocytes/field (median, range). M-234p: ( A ) CD4 + cells, N. S. ( B ) CD8 + cells, N. S. ( C ) Foxp3 + cells: Control vs Los, P < 0.05, vs Cy+Los, ( P < 0.05); ( D – F ) representative images of Control and Cy+Los treated tumors, 100× magnification; M-406: ( G ) CD4 + cells, N. S. ( H ) CD8 + cells, N. S. ( I ) Foxp3 + cells: Control vs Cy+Los, ( P < 0.05); ( J – L ) representative images of Control and Cy+Los treated tumors, 100× magnification. Kruskal-Wallis multiple comparison test and Dunn’s post-test.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: Quantification of tumor infiltrating lymphocytes by flow cytometry: M-234p, day 42, Cy vs Cy+Los: ( A ) CD4 cells, N. S. ( B ) CD8 cells, N. S. ( C ) Treg cells, ( P < 0.001). ( D ) Th17 cells, ( P < 0.05). Kruskal-Wallis multiple comparison test and Dunn’s post -test.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Flow Cytometry, Comparison

Journal: Oncotarget

Article Title: Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

doi: 10.18632/oncotarget.27694

Figure Lengend Snippet: αSMA : % of αSMA + area/field (median, range). ( A ) M-234p Control vs Los, ( P < 0.05), vs Cy+Los, ( P < 0.01). ( B ) M-406 Control vs Cy, ( P < 0.01), vs Los ( P < 0.01), vs Cy+Los ( P < 0.001). ( C ) M-234p and ( D ) M-406 representative images of Control and Cy+Los treated tumors, 100× magnification. Kruskal-Wallis multiple comparison test and Dunn’s post-test. Collagen : % of collagen area/field (median, range). ( E ) M-234p Control vs Cy+Los ( P < 0.01). ( F ) M-406 N. S. ( G ) M-234p and ( H ) M-406 representative images of Control and Cy+Los treated tumors, 100× magnification.

Article Snippet: Data obtained was analyzed using ANOVA and Tukey-Kramer Multiple Comparison tests, Kruskal-Wallis and Dunn’s post-test, and Log-rank tests were used to examine the differences between groups with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).

Techniques: Control, Comparison