Journal: Nature Communications

Article Title: Histone demethylase KDM2A is a selective vulnerability of cancers relying on alternative telomere maintenance

doi: 10.1038/s41467-023-37480-2

Figure Lengend Snippet: a Western blot analysis of ATRX protein expression in whole-cell lysates prepared from the indicated non-ALT and ALT-dependent (ALT+) cells. b Western blot analysis of ATRX expression in whole-cell lysates prepared from control or ATRX-depleted IMR90-T cells. The ATRX-depleted lines (dATRX clone#1–7) were established from clonally isolated IMR90-T cells transduced with ATRX-targeted sgRNA. The lysates from U2OS and ALT#1 cells were included as negative controls of ATRX expression. c Representative immuno-FISH images of 53BP1 and telomeres in ALT#1, IMR90-T (T#1), or ATRX-depleted IMR90-T (dATRX#1 and dATRX#2) cells. Scale bar, 10 μm. d Percentages of cells containing ≥4 53BP1-associated telomere dysfunction-induced foci (TIFs). Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t -test. e Competition-based proliferation assay of KDM2A-targeted sgK#1 (linked with GFP expression) in the indicated cell lines. Data were expressed as mean ± s.e.m. of three independent experiments. f Western blot analysis of Flag and ACTB (loading control) using whole-cell lysates prepared from control or Flag-tagged wild-type KDM2A-transduced HeLa, ALT#1, or Saos2 cells. g , h Telomere dot-blot analysis ( g ) and quantification ( h ) of anti-Flag or IgG chromatin immunoprecipitation (ChIP) in the indicated cell lines. The IgG ChIP was included as a control for non-specific signals. The input and ChIP DNAs processed against the indicated antibodies were assayed by dot-blotting and hybridized with a 32 P-labeled TelG probe. The relative enrichment was calculated after the normalization of ChIP DNA signals to the respective input DNA signals. Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t -test. i , j Telomere dot-blot analysis ( i ) and quantification ( j ) of anti-H3K36me2, anti-H3, or IgG ChIP in sgCtrl or sgK#1-transduced Saos2 cells. The relative enrichment was calculated after normalization to the respective ChIP DNA signals of sgCtrl-transduced samples. Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t -test. k Western blot analysis of KDM2A, H3K36me2, and H3 in cell lysates prepared from sgCtrl or sgK#1-transduced Saos2 cells. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were prepared in blocking solution as following dilutions: 53BP1 (1:500, IHC-00001; Bethyl Laboratories), 53BP1 (1:300, AF1877; R&D Systems), BLM (1:250, Cat# A300-110A; Bethyl Laboratories), Flag (1:200, F1804; Sigma), γH2AX (1:2,000, A300-081A; Bethyl Laboratories), phospho-Histone H3 (Ser10) (1:200, #9701; Cell Signaling), PML (1:200, sc-966; Santa Cruz Biotechnology), PML (1:500, ab96051; Abcam), SMC5 (1:400, A300-236A; Bethyl Laboratories), SUMO2/3 (1:250, ab3742; Abcam), and TRF1 (1:200, ab10579; Abcam).

Techniques: Western Blot, Expressing, Control, Isolation, Transduction, Two Tailed Test, Proliferation Assay, Dot Blot, Chromatin Immunoprecipitation, Labeling

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Influence of HO-1 induction on p38 MAPK activation in BeWo cells under T. gondii infection. (A,B) BeWo (1 × 10 6 cells/2,000 μl/well) cells were cultured in six-well plate during 24 h, infected with T. gondii (1:1) or not (control) for 3 h, treated with hemin (80 μM) or not (NaOH at 0.08% as vehicle) during 24 h, and submitted to western blotting assays for detection of phosphorylated p38 MAPK and β-actin. Protein bands were photodocumented, and densitometric analysis was performed by the ratio among phosphorylated p38 MAPK ( p -p38 MAPK) and its corresponding β-actin (endogenous control). (A) Representative blots for p -p38 MAPK and β-actin in the non-infected (NI) or infected ( T. gondii ) and hemin-treated or not (vehicle) BeWo cells. (B) Densitometric analysis for p -p38 MAPK. (C) BeWo (3 × 10 4 cells/200 μl/well) cells were cultured in 96-well plate during 24 h, pretreated with 10 μM of SB203580 (p38 MAPK inhibitor) or not (control) for 3 h, infected with T. gondii (1:1) for 3 h, and treated with hemin (80 μM) or not (control) during 24 h, and the T. gondii intracellular proliferation was analyzed by the β-galactosidase assay. The data were shown as number of tachyzoites (× 10 3 ). For both assays, the results were expressed as mean ± SEM from two or three independent experiments performed in six or eight replicates. Significant differences were statistically assessed by the Mann-Whitney test (B) or by one-way ANOVA with Bonferroni’s posttest (C) ; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Activation Assay, Infection, Cell Culture, Control, Western Blot, MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Proposed mechanisms triggered by the HO-1 to control T. gondii infection by BeWo trophoblast cells. (A) Upper panel, a non-infected BeWo cell expresses normal levels of HO-1 and phosphorylated p38 MAPK, in addition to secreting baseline levels of IL-6. (B) Left panel, when BeWo cells were infected with T. gondii , the amounts of HO-1 and phosphorylated p38 MAPK were significantly decreased and the IL-6 levels were upregulated. (C) Right panel, when finally T. gondii -infected BeWo cells were treated with hemin, a specific HO-1 inducer, the parasite proliferation was significantly reduced, sequentially associated with an overexpression of HO-1, an increasing of the p38 MAPK phosphorylation and a releasing of high IL-6 levels, which was higher than those naturally secreted by the T. gondii -infected cells non-induced for HO-1 (left panel).

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Control, Infection, Over Expression, Phospho-proteomics