Journal: Frontiers in Immunology

Article Title: Gut Microflora Modulates Th17/Treg Cell Differentiation in Experimental Autoimmune Prostatitis via the Short-Chain Fatty Acid Propionate

doi: 10.3389/fimmu.2022.915218

Figure Lengend Snippet: Evaluation of the EAP model, proportion of Th17/Treg cells, and levels of related inflammatory cytokines. (A) After 1 week of acclimation, mice received primary and secondary immunization on days 0 and 15, respectively. Modeling was completed 1 week later, followed by feces collection and pain measurement for 2 days. Mice were sacrificed on day 24 and then samples were collected. (B) Representative HE staining images of prostate tissue sections of the control and EAP group. The black arrowhead indicates infiltration of inflammatory cells in the EAP group. (C) Inflammation score of prostate in the control and EAP group. (D) EAP induced pelvic pain as evaluated by tactile allodynia. (E) Representative pictures of flow cytometric staining for CD4 + IL-17 + cells in the splenic lymphocytes from control and EAP mice. (F) Flow cytometric analysis of the proportion of CD4 + IL-17 + cells in control and EAP mice. (G) Representative pictures of flow cytometric staining for CD4 + RORγt + cells in the splenic lymphocytes from control and EAP mice. (H) Flow cytometric analysis of the proportion of CD4 + RORγt + cells in control and EAP mice. (I) Representative pictures of flow cytometric staining for CD4 + CD25 + FoxP3 + cells in the splenic lymphocytes from control and EAP mice. (J) Flow cytometric analysis of the proportion of CD4 + CD25 + FoxP3 + cells in control and EAP mice. (K) Serum concentrations of IL-17, TGF-β, IL-10, GM-CSF, and IFNγ by ELISA in the control and EAP group. (L) Concentrations of IL-17, TGF-β, and IL-10 in 10-mg prostate tissues in the control and EAP group. (M) Immunohistochemical staining of IL-17A (marker of Th17) on prostate sections in the control and EAP group. (N) Immunohistochemical staining of FoxP3 (marker of Treg) on prostate sections in the control and EAP group. The black arrows indicate the IL-17/FoxP3-positive cells in the two groups. Data are from one experiment representative of three independent experiments. n = 5–6/group; * p < 0.05; ** p < 0.01; EAP, experimental autoimmune prostatitis; HE, hematoxylin–eosin; IL-17, interleukin-17; RORγt, retinoic acid−related orphan receptor γt; GM-CSF, granulocyte-macrophage colony stimulating factor; IFNγ, interferon gamma; TGF-β, transforming growth factor-β; IL-10, interleukin-10; FoxP3, forkhead box P3; Treg, regulatory T cell.

Article Snippet: Next, cells were fixed and permeated successively, followed by incubating with intracellular antibody, including PE-labeled IL-17A (rat anti-mouse, BD, 559502, USA), PE-labeled retinoic acid−related orphan receptor γt (RORγt, rat anti-mouse, ebioscience, 12-6981-60, USA), and PE-labeled FoxP3 (rat anti-mouse, BD, 563101, USA), at a temperature of 4°C for 60 min. After being washed again and analyzed by a FACSCalibur flow cytometer (Beckman Coulter, USA), the resulting data were processed by FlowJo software (Tree Star, Ashland).

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Marker

Journal: Frontiers in Immunology

Article Title: Gut Microflora Modulates Th17/Treg Cell Differentiation in Experimental Autoimmune Prostatitis via the Short-Chain Fatty Acid Propionate

doi: 10.3389/fimmu.2022.915218

Figure Lengend Snippet: Propionic acid ameliorates EAP and restores Th17/Treg imbalance in vivo. (A) After 1 week of acclimation, all mice received EAP induction. They were divided into an untreated group (EAP group) and a propionate-treated group (EAP+PA group) according to whether propionic acid was added in drinking water or not. Mice were sacrificed on day 24 and then samples were collected. (B) Representative HE staining of prostate sections from untreated and propionate-treated EAP mice. The black arrowhead indicates infiltration of inflammatory cells in the EAP group. (C) Inflammation score of prostate sections from untreated and propionate-treated EAP mice. (D) Chronic pelvic pain measurement of untreated and propionate-treated EAP mice. (E) Representative pictures of flow cytometric staining for CD4 + IL-17 + cells in the splenic lymphocytes from untreated and propionate-treated EAP mice. (F) Flow cytometric analysis of the proportion of CD4 + IL-17 + cells in untreated and propionate-treated EAP mice. (G) Representative pictures of flow cytometric staining for CD4 + RORγt + cells in the splenic lymphocytes from untreated and propionate-treated EAP mice. (H) Flow cytometric analysis of the proportion of CD4 + RORγt + cells in untreated and propionate-treated EAP mice. (I) Representative pictures of flow cytometric staining for CD4 + CD25 + FoxP3 + cells in the splenic lymphocytes from untreated and propionate-treated EAP mice. (J) Flow cytometric analysis of the proportion of CD4 + CD25 + FoxP3 + cells in untreated and propionate-treated EAP mice. (K) Detection of serum cytokine productions (IL-17, GM-CSF, IFNγ, TGF-β, and IL-10) from untreated and propionate-treated EAP mice. (L) Concentration determination of IL-17, TGF-β, and IL-10 in 10 mg of prostate tissue in untreated and propionate-treated EAP mice. (M) Immunohistochemical staining of IL-17A (marker of Th17) on prostate sections in untreated and propionate-treated EAP mice. (N) Immunohistochemical staining of FoxP3 (marker of Treg) on prostate sections in untreated and propionate-treated EAP mice. The black arrows indicate the IL-17/FoxP3-positive cells in the two groups. Data are from one experiment representative of three independent experiments. Data are representative of three independent experiments. n = 5–6/group; * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant; EAP, experimental autoimmune prostatitis; HE, hematoxylin–eosin, PA, propionic acid; IL-17, interleukin-17; RORγt, retinoic acid−related orphan receptor γt; GM-CSF, granulocyte-macrophage colony stimulating factor; IFNγ, interferon gamma; TGF-β, transforming growth factor-β; IL-10, interleukin-10; FoxP3, forkhead box P3; Treg, regulatory T cell.

Article Snippet: Next, cells were fixed and permeated successively, followed by incubating with intracellular antibody, including PE-labeled IL-17A (rat anti-mouse, BD, 559502, USA), PE-labeled retinoic acid−related orphan receptor γt (RORγt, rat anti-mouse, ebioscience, 12-6981-60, USA), and PE-labeled FoxP3 (rat anti-mouse, BD, 563101, USA), at a temperature of 4°C for 60 min. After being washed again and analyzed by a FACSCalibur flow cytometer (Beckman Coulter, USA), the resulting data were processed by FlowJo software (Tree Star, Ashland).

Techniques: In Vivo, Staining, Concentration Assay, Immunohistochemical staining, Marker

Journal: Breast Cancer Research and Treatment

Article Title: Targeting ataxia telangiectasia-mutated- and Rad3-related kinase (ATR) in PTEN-deficient breast cancers for personalized therapy

doi: 10.1007/s10549-018-4683-4

Figure Lengend Snippet: Association between PTEN, ATR and pChk1expression in breast cancers

Article Snippet: A further set of slides were incubated for 60 min with the primary rabbit polyclonal anti-phosphorylated Chk1 antibody (Ab58567, Abcam, Cambridge, UK), at a dilution of 1:140 [ ].

Techniques:

Journal: Breast Cancer Research and Treatment

Article Title: Targeting ataxia telangiectasia-mutated- and Rad3-related kinase (ATR) in PTEN-deficient breast cancers for personalized therapy

doi: 10.1007/s10549-018-4683-4

Figure Lengend Snippet: a Nuclear and cytoplasmic staining of PTEN [a negative staining for both, b negative staining for nuclei and weak for cytoplasm, c some weak staining for nuclei and moderate for cytoplasm d strong staining for both; TMA cores pictures were taken using digital pathology interference at ×100 (left) and ×200 (right)]. Kaplan–Meier plot showing breast cancer-specific survival (BCSS) and b nuclear PTEN level. c combined nuclear PTEN and ATR level. d combined nuclear PTEN and cytoplasmic pCHK1 level. e combined ATR and cytoplasmic pCHK1 level nuclear PTEN negative tumours

Article Snippet: A further set of slides were incubated for 60 min with the primary rabbit polyclonal anti-phosphorylated Chk1 antibody (Ab58567, Abcam, Cambridge, UK), at a dilution of 1:140 [ ].

Techniques: Staining, Negative Staining