Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Growing Kit 225 cells were transfected with RelA-specific siRNA and cultured for 72 h. RelA expression was examined by RT-PCR. GAPDH was used as an internal control. (B) Growing Kit 225 cells were transfected with RelA-specific siRNA 24 h before adenovirus infection (Ad-Tax1 or Ad-Con), and harvested 48 h and 72 h post infection for western blotting. RelA, p100, p52 and Tax1 expression was monitored by immunoblotting with anti-RelA, anti-p52 and anti-Tax1 antibodies. β-Tubulin was used as an internal control. (C) siRNA-treated growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 to Ad-Con are shown. *, p <0.05. (D) siRNA-treated growing Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for 72 h. Tax1 expression and DNA fragmentation were measured by flow cytometry. (E) Percentage average number of cells undergoing apoptosis was calculated from three independent experiments. Values are shown as the means ± SE. *, p < 0.05.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Infection, Western Blot, Activity Assay, MTT Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Kit 225 cells were transfected with p100-specific siRNA and cultured for 48 h. p100 expression was detected by western blotting. β-Tubulin was used as an internal control. (B) Growing Kit 225 cells were transfected with p100-specific siRNA and cultured for 24 h, and infected with Ad-Con or Ad-Tax1, followed by harvest at indicated times. Mitochondrial activity was measured by MTT assay. Relative values of Ad-Tax1 to Ad-Con are shown. *, p < 0.05. (C) Expression of adenovirus-derived Tax1 and its mutant Tax225–232 proteins was measured by immunoblotting with anti-Tax1 antibody (Taxy-7). p100 and p52 levels were detected by anti-p52 antibody, which recognizes both p100 and p52. β-Tubulin was used as an internal control. (D) Growing Kit 225 cells were infected with Ad-Tax1, its mutant Ad-Tax225–232 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 or Ad-Tax225–232 samples to Ad-Con samples are shown. *, p < 0.05. (E) Growing Kit 225 cells were infected with Ad-Tax1 or Ad-Tax225–232, and cultured for 72 h. DNA fragmentation was measured by flow cytometry. The thick line and gray area indicate Ad-Tax1- or Ad-Tax225–232-treated cells and Ad-Con-treated cells, respectively.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Western Blot, Control, Infection, Activity Assay, MTT Assay, Derivative Assay, Mutagenesis, Flow Cytometry

Journal: Nature

Article Title: Microbiota-targeted maternal antibodies protect neonates from enteric infection

doi: 10.1038/s41586-019-1898-4

Figure Lengend Snippet: a, Bacterial burdens of reciprocally bred mNab+ and mNab− pups (6–7 days old) orally challenged with 107 CFU of ETEC 6. Ig. Immunoglobulin; SI, small intestine. **P = 0.0004, two-tailed Mann–Whitney U-test. Data are representative of four independent experiments. b, Survival among reciprocally bred pups 20 h after oral–gastric challenge with 109 CFU of ETEC 6. Data are from three independent experiments (first experiment, n = 8 mNab+ mice, n = 5 mNab− mice; second experiment, n = 9 mNab+ mice, n = 9 mNab− mice; third experiment, n = 7 mNab+ mice, n = 6 mNab− mice). *P = 0.0011, two-tailed unpaired t-test. c, Serum IgG levels in ETEC-challenged reciprocally bred pups. ***P = 0.0002, two-tailed Mann–Whitney U-test. Data are representative of two independent experiments. d, Small-intestinal mucosal IgG levels in ETEC-challenged reciprocally bred pups. **P = 0.0022, two-tailed Mann–Whitney U-test. Data are representative of two independent experiments. SI, small intestine. e, Flow cytometry analysis of natural maternal IgG and IgA coating of commensal bacteria of 1-week-old mNab+ and mNab− pups. Data are representative of two independent experiments (n = 4–5 mice per group in each experiment). f, Flow cytometry analysis of natural maternal IgG and IgA coating of ETEC–GFP bacteria in mNab+ and mNab− pups 18 h after infection. IgG and IgA signals are gated on GFP+ population. Data are representative of two independent experiments (n = 4–7 mice per group in each experiment). g, Serum IgG levels after 1 week of cross-fostering. h, Small-intestinal IgG levels after 1 week of cross-fostering. i, Small-intestinal IgA levels after 1 week of cross-fostering. j, ETEC 6 bacterial burdens in the small intestine of pups cross-fostered for 1 week. ***P = 0.0002, two-tailed Mann–Whitney U-test. Data are representative of two independent experiments. a–d, g–j, Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Article Snippet: Isotype controls were Alexa Fluor 647 goat IgG (Biolegend, 403006) and rat IgG1–APC (SouthernBiotech, 0116–11).

Techniques: Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Infection

Journal: Nature

Article Title: Microbiota-targeted maternal antibodies protect neonates from enteric infection

doi: 10.1038/s41586-019-1898-4

Figure Lengend Snippet: a, Breeding and fostering strategy to specifically study the postnatal milk IgG transfer process. All pups discussed in this figure are μMT−/−. b, Serum IgG levels in 1-week-old FcRn-deficient or FcRn-sufficient μMT−/− pups after 1 week of fostering by a μMT+/− dam. **P = 0.0013, two-tailed Mann–Whitney U-test. Data are representative of two independent experiments. c, Titres in pups of IgG specific to the microbiota of the foster dam. Data are representative of two independent experiments. d, Adult (8-week-old) mice were intraperitoneally (i.p.) injected with 5 mg of IgG, and faeces samples were collected 1 day later. Faecal IgG levels are shown as μg per gram of faeces. *P = 0.0357, two-sided Mann–Whitney U-test. Data are representative of two independent experiments. e, IgG treatment scheme of dams treated with IgG, data for the pups were tested are shown in f and g. f, Comparison of ETEC 6 bacterial burden in the small intestine of pups from untreated μMT−/− dams and from μMT−/− dams treated with IgG. **P = 0.0043, two-sided Mann–Whitney U-test. Data are representative of two independent experiments. g, Serum IgG levels of pups from untreated μMT−/− dams compared with μMT−/− dams treated with IgG. Data are representative of two independent experiments. b–d, f, g, Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Article Snippet: Isotype controls were Alexa Fluor 647 goat IgG (Biolegend, 403006) and rat IgG1–APC (SouthernBiotech, 0116–11).

Techniques: Two Tailed Test, MANN-WHITNEY, Injection

Journal: Nature

Article Title: Microbiota-targeted maternal antibodies protect neonates from enteric infection

doi: 10.1038/s41586-019-1898-4

Figure Lengend Snippet: a, Total immunoglobulin titres against ETEC 6 in serum from germ-free (GF) and SPF adult female mice as well as in serum from SPF mice absorbed by mouse microbiota. Data are representative of four independent experiments. OD405, optical density at 405 nm. b, IgG titres against ETEC 6 in serum from germ-free and SPF mice. c, Total immunoglobulin titres against ETEC 6 in serum from 1-week-old neonatal mNab+ and mNab− mice obtained by reciprocal breeding. d, IgG titres against ETEC 6 in serum from 1-week-old neonatal mNab+ and mNab− mice obtained by reciprocal breeding. e, Total immunoglobulin titres against ETEC 6 in serum from germ-free mice, serum from SPF mice and Pantoea-1-absorbed serum from SPF mice. f, IgG titres against ETEC 6 in serum from germ-free mice, serum from SPF mice and Pantoea-1-absorbed serum from SPF mice. Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Article Snippet: Isotype controls were Alexa Fluor 647 goat IgG (Biolegend, 403006) and rat IgG1–APC (SouthernBiotech, 0116–11).

Techniques:

Journal: Nature

Article Title: Microbiota-targeted maternal antibodies protect neonates from enteric infection

doi: 10.1038/s41586-019-1898-4

Figure Lengend Snippet: a, Survival of pups born to ETEC-6- or Pantoea-1-immunized dams or unimmunized dams. Data are from two individual experiments (first experiment, ETEC n = 12 mice, unimmunized (non) n = 7 mice, Pantoea n = 4 mice; second experiment, ETEC n = 5 mice, unimmunized n = 5 mice, Pantoae n = 4 mice). b, Liver total bacterial burdens 3 days after intraperitoneal ETEC 6 challenge. **P = 0.0025, one-way analysis of variance (ANOVA) with Bonferroni post-test. Data are from two independent experiments. c, Spleen bacterial burdens 3 days after intraperitoneal ETEC 6 challenge. **P = 0.0041, one-way ANOVA with Bonferroni post-test. Data are from two independent experiments. d, Cross-reactivity against ETEC 6 of serum IgG from pups born to germ-free dams with or without Pantoea 1 immunization. e, Western blot showing that serum IgG of pups born to a Pantoea-1-immunized dam recognizes antigens in cellular lysates of members of the Enterobacteriaceae family (ETEC 6, Pantoea 1 and Enterobacter). Lane 1, Staphylococcus; lane 2, ETEC 6; lane 3, Pantoea 1; lane 4, Enterobacter. Blot is detected with goat anti-mouse IgG antibody. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. b–d, Data are mean ± s.e.m.

Article Snippet: Isotype controls were Alexa Fluor 647 goat IgG (Biolegend, 403006) and rat IgG1–APC (SouthernBiotech, 0116–11).

Techniques: Western Blot