Journal: PLoS ONE

Article Title: Intestinal Intraepithelial Lymphocyte-Enterocyte Crosstalk Regulates Production of Bactericidal Angiogenin 4 by Paneth Cells upon Microbial Challenge

doi: 10.1371/journal.pone.0084553

Figure Lengend Snippet: (A) iIELs from WT and TCRδ ‑/‑ mice were cultured at 37°C for 6h with medium alone or medium containing 1x10 5 CFU S . Typhimurium or recombinant IL‑23 (100ng/ml) after which supernatants were analysed by IL‑22 ELISA (*p≤0.05). Data (mean±SEM) were collated from three experiments. (B) Intestinal epithelial cells (10 6 ) from WT and TCRδ ‑/- mice were exposed for 2h to medium alone or medium containing Salmonella at a ratio of 10 bacteria per epithelial cell. IL‑23 mRNA expression was then assessed by qPCR. The data (mean±SEM) were expressed relative to mRNA levels found in WT epithelial cells exposed to medium alone and were collated from three experiments (*p≤0.05). (C) m-ICcl2 intestinal epithelial cells were cultivated on 0.4µm pore size filters and incubated at 37°C for 4h with medium alone, or medium containing invasive or non‑invasive Salmonella after which cells were harvested, RNA extracted and IL‑23 expression analysed by qPCR. Data are expressed relative to mRNA levels obtained on m‑ICc12 cells exposed to medium alone. (D) RNA was purified from lamina propria (LP) cells isolated from orally infected (2h p.i.) and non‑infected WT mice. As a positive control for IL-23 production, cultivated RAW264.7 macrophages (RAW) were analysed pre- (NI) and post-infection (I) (6h p.i.) with Salmonella . Data are shown as mean±STD relative to mRNA levels in non‑infected samples, and are representative of 5 and 4 experiments, respectively. (E) iIELs from WT mice were cultured at 37°C for 2h with medium alone (No Stimulus) or with medium containing IL‑23. The distribution of IL-23Rα expression among Vγ1 and Vγ7 iIELs was determined by flow cytometry from 4-colour/antibody staining protocols: combining CD3, TCR-γδ, IL-23R and either TCR-Vγ7 or TCR-Vγ1 antibodies with a gating strategy of selecting CD3 + , TCR-γδ + , IL-23R + events (middle panels) and then analysing them for Vγ7 + , IL-23Rα + and Vγ7 - , IL-23Rα + events (far right hand panels). The profiles shown are representative of three experiments with the percentage values representing the frequency of positive cells in the designated quadrants.

Article Snippet: In vitro control experiments were carried out on the mouse trans‐immortalised cell line of intestine epithelial cells, m‐ICc12 [ ] and on the mouse tumour‐derived macrophages, RAW264.7 (ATCC® TIB71TM), cultivated to a density of 1 and 6-8x10 6 cells, respectively , in T25 flasks and infected with S . Typhimurium SL1344 at a multiplicity of infection of 10:1 and 100:1, respectively [ , , ] or with gut commensal isolates grown overnight and used at a multiplicity of infection of 10:1.

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Bacteria, Expressing, Pore Size, Incubation, Purification, Isolation, Infection, Positive Control, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Intestinal Intraepithelial Lymphocyte-Enterocyte Crosstalk Regulates Production of Bactericidal Angiogenin 4 by Paneth Cells upon Microbial Challenge

doi: 10.1371/journal.pone.0084553

Figure Lengend Snippet: (A) Invasive or non-invasive Salmonella strains (4x10 7 CFU in PBS) were injected into exteriorised intestinal ligated loops of WT mice. Four hours later mucosal RNA was isolated and Ang4 mRNA expression analysed by qPCR. The data (mean±SEM) are expressed relative to mRNA levels obtained from loops exposed to PBS alone (n=3; ***p<0.001). (B) IL‑23 protein levels assessed by ELISA (mean±SEM) from intestinal epithelial cells of WT mice exposed for 12h to medium alone, (+Medium) or media containing invasive or non‑invasive Salmonella strains at a ratio of 10 bacteria per epithelial cell (n=3; ***p<0.001, *p<0.05). (C) m-ICc12 intestinal epithelial cells were cultured for 4h with medium alone (control) or containing live cells of various intestinal commensal bacteria at a ratio of 10 bacteria per epithelial cell. IL‑23 mRNA expression was assessed by qPCR. Data (mean±SEM) were expressed relatively to mRNA levels in control samples (n=4). (D) IL‑23 protein production measured by ELISA in intestinal epithelial cells from WT mice cultured at 37°C for 12h with medium alone or containing either, live WT (SL1344) or various Salmonella mutant strains that are non-invasive (SL1344ΔSPI1), invasive but non-flagellated (JH3220=SL1344Δ fliC Δ fljB ), non-invasive and non-flagellated (JH3515=SL1344∆SPI1Δ fliC Δ fljB ) or non-invasive, flagellated but unable to transcytose flagellin (JH3574=SL1344∆SPI1Δ ssrA ) (n=3; mean±SEM). (E) IL‑23 mRNA expression assessed by qPCR in m-ICc12 epithelial cells cultured at 37°C for 4h with medium alone or containing live or heat‑killed WT Salmonella SL1344 cells at a ratio of 10 bacteria per epithelial cell. Data (n=4; mean±SEM) are expressed relative to mRNA levels obtained in non‑infected cells. (F) IL‑23 mRNA levels analysed by qPCR in intestinal epithelial cells from WT mice cultured at 37°C for 2h with medium alone or containing lipopolysaccharide (LPS), peptidoglycan (PGN), muramyl dipeptide (MDP) or methylated DNA (CpG). Data (n=3; mean±SEM) are expressed relative to mRNA levels obtained in cells exposed to medium alone.

Article Snippet: In vitro control experiments were carried out on the mouse trans‐immortalised cell line of intestine epithelial cells, m‐ICc12 [ ] and on the mouse tumour‐derived macrophages, RAW264.7 (ATCC® TIB71TM), cultivated to a density of 1 and 6-8x10 6 cells, respectively , in T25 flasks and infected with S . Typhimurium SL1344 at a multiplicity of infection of 10:1 and 100:1, respectively [ , , ] or with gut commensal isolates grown overnight and used at a multiplicity of infection of 10:1.

Techniques: Injection, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Bacteria, Cell Culture, Control, Mutagenesis, Methylation

Journal: PLoS ONE

Article Title: Intestinal Intraepithelial Lymphocyte-Enterocyte Crosstalk Regulates Production of Bactericidal Angiogenin 4 by Paneth Cells upon Microbial Challenge

doi: 10.1371/journal.pone.0084553

Figure Lengend Snippet: (1) Upon exposure to Salmonella or recognition of commensal bacteria or MAMPs, intestinal epithelial cells secrete IL‑23, in a TLR‑dependent manner in the case of Salmonella . (2) Via extracellular or transcellular routes, epithelial cells secrete IL‑23 (pink to grey gradient arrows) that binds to its cognate receptor IL‑23R expressed by γδ iIELs. (3) IL-23R + iIELs enriched in Vγ7 + cells respond to IL-23 by secreting IL‑22 (yellow to grey gradient arrows). Via extracellular or transcellular routes IL‑22 acts on IL-22R-bearing Paneth cells up-regulating Ang4 transcription (4) and/or secretion (5) of pre-formed protein stored in intracellular granules. Ang4 is secreted into the lumen at levels sufficient (5) to effectively kill Salmonella located in the vicinity of the intestinal tissue (6), helping to protect it from proliferation of and invasion by the pathogen.

Article Snippet: In vitro control experiments were carried out on the mouse trans‐immortalised cell line of intestine epithelial cells, m‐ICc12 [ ] and on the mouse tumour‐derived macrophages, RAW264.7 (ATCC® TIB71TM), cultivated to a density of 1 and 6-8x10 6 cells, respectively , in T25 flasks and infected with S . Typhimurium SL1344 at a multiplicity of infection of 10:1 and 100:1, respectively [ , , ] or with gut commensal isolates grown overnight and used at a multiplicity of infection of 10:1.

Techniques: Bacteria

Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A) Venn diagram of the 18 candidate genes shared by the Macro 1, Strong, and Positive subgroups. (B) Violin plot of the CD74, GRN, CYBA, and CTSH expression in the Strong, Moderate, and Weak subgroups. (C) The normalized CD74 expression level after pre-treatment, NACT, and anti-PD-1 Ab combination therapy (NACT+anti-PD-1 Ab) in T2, T3, and T4 patients. The patient T3 samples received two rounds of NACT and anti-PD-1 Ab combination therapy were analyzed. (D) UMAP projection of the CD74 expression in macrophage subgroups. The expression plot was split based on the Strong, Moderate, and Weak subgroups. (E) The unsupervised transcriptional trajectory of macrophages from Monocle (version 3), colored by the pseudo-time. The circle labeled 1 indicated the start of the trajectory. (F) UMAP projection of the normalized expression of CD74, GRN, NPC2, CYBA, CTSH, and LAPTM5 in macrophage subgroups. The gray curve indicates the pseudo-time trajectory. (G) The CD74 and GRN expression dynamic change along with the pseudo-time, colored by the Strong, Moderate, and Weak subgroups. (H) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression in the tissues of patient T4 receiving NACT and anti-PD-1 Ab combination therapy. The green boxes represent CD74 negative macrophages, and the white boxes represent macrophages expressing CD74. (I) The CD74 mRNA level in HeLa or CaSki cells cocultured with macrophages derived from THP-1 cells. Anti-PD-1 Ab and CDDP were used to establish a model of NACT combined with immunotherapy in vitro. (J) The percentage of CD74-positive macrophages cocultured with CaSki cells was detected by flow cytometry. (K) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression of subcutaneous tumors treated with CDDP and/or anti-PD-1 Ab. (L) Flow cytometry revealed the number of CD74-positive macrophages in single-cell suspensions isolated from mice subcutaneous tumors. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD.

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Expressing, Labeling, Immunofluorescence, Derivative Assay, In Vitro, Flow Cytometry, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A, B) Phagocytosis ability detected by flow cytometry. Macrophages derived from THP-1 cells were co-cultured with SiHa cells knockdown (A) or overexpressing (B) CD74. Phagocytosis efficiency was quantified by the percentage of double fluorophore-positive macrophages in the upper right quadrant. (C) The phagocytic function of macrophages co-cultured with SiHa cells treated with anti-CD74 was measured by flow cytometry. (D) Density plot of the M1 and M2 ssGSEA score in the Strong, Moderate, and Weak subgroups. (E) M1/M2 ssGSEA ratio of the macrophages in the pretreatment, NACT, and anti-PD-1 Ab combination group. (F, G) UMAP projection of the normalized density of CD74 expression (F), M1 score, and M2 score (G) in the macrophage subgroups. (H, I) CD86 (H) and CD206 (I) expression of THP-1-derived macrophages after CD74 overexpression. CD86 is the surface marker of M1 macrophages, while CD206 is the surface marker of M2 macrophages. (J, K) CD86 (J) and CD206 (K) expression of THP-1-derived macrophages after CD74 knockdown. (L) Phagocytosis efficiency of THP-1-derived macrophages cocultured with SiHa cells. CDDP-induced phagocytic function loss of macrophages was rescued by knocking down CD74. (M) Flow cytometry detected the differences in phagocytic function among the control, the CDDP, and the anti-CD74 Ab combined with the CDDP group. THP-1-derived macrophages were cocultured with SiHa cells. (N) The representative image of the subcutaneous tumor model. Mouse cervical cancer cells TC-1 were subcutaneously inoculated into immunocompetent CD74 humanized BALB/c-hCD74 mice. Each group consisted of 6 mice receiving CDDP or CDDP combined with intraperitoneal injected anti-CD74 Ab. Tumors of four mice in the drug-combination group became nonpalpable on Day 4 after administration. (O) Growth curve of subcutaneous tumors in mice. (P) Phagocytosis assay was performed using macrophages isolated from the mice’s spleen and mouse cervical cancer cell line TC-1. Phagocytosis efficiency was quantified by the percentage of Dil and Dio double fluorophore-positive THP-1-derived macrophages. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD. *p<0.05, **p < 0.01, ***p<0.001

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Knockdown, Expressing, Over Expression, Marker, Control, Injection, Phagocytosis Assay, Isolation, Two Tailed Test

Journal: International journal of biological sciences

Article Title: Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

doi: 10.7150/ijbs.45710

Figure Lengend Snippet: Figure 3. Dioscin induced cell apoptosis in lung SCC cells. NCI‐H520, SK‐MES‐1 and HBE cells were treated with dioscin (0, 1.25, 2.5, 5 µM) for 48 h. (A, B) Cell apoptosis was measured by flow cytometry. (C) The changes in ΔΨm were monitored by JC-1 staining and the ratio of green/red fluorescence intensity was used to calculate mitochondrial depolarization. Data are the mean ± SD of triplicate samples. Significant differences compared with the control are indicated by *p<0.05, **p<0.01, and***p<0.001. (D) The expression levels of cleaved caspase-3, cleaved PARP, Bax and Bcl-2 were analysed by western blot.

Article Snippet: Antibodies against Bcl2, Bax, cleaved caspase-3, cleaved PARP, p38, p-p38 (Thr180/ Tyr182), E-cadherin, N-cadherin and Vimentin were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Flow Cytometry, Staining, Fluorescence, Control, Expressing, Western Blot

Journal: International journal of biological sciences

Article Title: Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

doi: 10.7150/ijbs.45710

Figure Lengend Snippet: Figure 4. The p38-MAPK/HSP27 signalling pathway was involved in dioscin-induced apoptosis. (A) NCI‐H520 and SK‐MES‐1 cells were treated with dioscin (5 µM) for 48 h. The levels of p-p38, p38, p-HSP27 and HSP27 were analysed by western blotting. (B, C) NCI‐H520 and SK‐MES‐1 cells were treated with Anisomycin (0.5 µM) for 24 h. Then, cell apoptosis was measured by flow cytometry, and the expression levels of p-p38, p38, p-HSP27, HSP27, Bax, Bcl2, cleaved caspase-3 and cleaved PARP were analysed by western blotting. (D) NCI‐H520 and SK‐MES‐1 cells were pretreated with SB203580 (5 µM) for 2 h before exposure to dioscin for 48 h. Then, cell apoptosis was measured by flow cytometry. Data are presented as the mean ± SD of triplicate samples. *P < 0.05, **P < 0.01, and ***P < 0.001 vs control.

Article Snippet: Antibodies against Bcl2, Bax, cleaved caspase-3, cleaved PARP, p38, p-p38 (Thr180/ Tyr182), E-cadherin, N-cadherin and Vimentin were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Western Blot, Flow Cytometry, Expressing, Control

Journal: International journal of biological sciences

Article Title: Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

doi: 10.7150/ijbs.45710

Figure Lengend Snippet: Figure 5. Intracellular ROS accumulation was a pivotal event in dioscin-induced apoptosis. (A, B) NCI‐H520 and SK‐MES‐1 cells were treated with dioscin (5 µM) for 48 h. The intracellular ROS levels were measured by flow cytometry. Data are presented as the mean ± SD of triplicate samples. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control. (C-H) NCI‐H520 and SK‐MES‐1 cells were pretreated with NAC (2 mM) for 24 h before exposure to dioscin for 48 h. Then, cell apoptosis and intracellular ROS levels were measured by flow cytometry. Data are presented as the mean ± SD of triplicate samples. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control. The expression levels of cleaved caspase 3, cleaved PARP, Bax, Bcl2, p-p38, p38, p-HSP27 and HSP27 were analysed by western blotting.

Article Snippet: Antibodies against Bcl2, Bax, cleaved caspase-3, cleaved PARP, p38, p-p38 (Thr180/ Tyr182), E-cadherin, N-cadherin and Vimentin were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Flow Cytometry, Control, Expressing, Western Blot

Journal: International journal of biological sciences

Article Title: Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

doi: 10.7150/ijbs.45710

Figure Lengend Snippet: Figure 6. In vivo anti-tumour effects of dioscin in xenograft models. NCI-H520 cells were subcutaneously injected into the backs of BALB/c nude mice. When the tumour reached a volume of 50-100 mm3, mice were divided into two groups and orally treated with vehicle or dioscin (80 mg/kg/d) for 12 days (n=6). (A) The tumour volumes of the mice were determined every three days after the onset of treatment. (B, C) On day 12, the tumours were carefully dissected from the mice and the weights of tumours was measured. (D, E) Apoptotic cells in tumour samples were detected by TUNEL assay (scale bar=20 µm). Data are presented as the mean ± SD. Significant differences compared with the control are indicated by *p<0.05, **p<0.01, and***p<0.001. (F) The harvested tumours were subsequently lysed and western blot analysis was performed for cleaved caspase-3, cleaved PARP, Bax, Bcl2, p-p38, p38, p-HSP27, HSP27 and GAPDH expression.

Article Snippet: Antibodies against Bcl2, Bax, cleaved caspase-3, cleaved PARP, p38, p-p38 (Thr180/ Tyr182), E-cadherin, N-cadherin and Vimentin were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: In Vivo, Injection, TUNEL Assay, Control, Western Blot, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Novel Role of p53 in Septic Immunosuppression: Involvement in Loss and Dysfunction of CD4+ T Lymphocytes.

doi: 10.1159/000495241

Figure Lengend Snippet: Fig. 4. p53 mediated the apoptosis of CD4+ T lymphocytes during sepsis. (A-C) Splenic CD4+ T cells were isolated from WT mice (n=20) and subjected to LPS (1 μg/ml) stimulation with or without PFT. After indicated time points, cell proteins were extracted and subjected to analysis of Bcl-2, Bax and cleaved caspase-3. β-actin was used as loading control (A). Cell proteins were subjected to analysis of caspase-3 activities. The original values were shown as means ± SD. *Significant difference when compared with 0 group (P<0.05). #Significant difference when compared with the PFT group (P<0.05) (B). Cells were stained with Annexin-V/7-ADD and then analyzed by flow cytometry (C). (D, E) WT mice (n=20 for each group) were subjected to CLP with or without a 30 min prior intraperitoneal injection of PFT (D). WT (n=10) or p53-/- mice (n=10) were subjected to CLP (E). Mice were sacrificed at 24 hours and the splenic CD4+ T cells were isolated. Cells were stained with Annexin-V/7-ADD and then analyzed by flow cytometry. Annexin+/7- AAD- indicated early apoptosis.

Article Snippet: Anti-phospho-p53 (Ser15) antibody, anti-phospho-mitogen-activated protein kinase (MAPK) (42/44) antibody, anti-Bcl-2 antibody, anti-Bax antibody and anti-cleaved caspase-3 antibody were purchased from Cell Signaling Technology Inc., Danvers, MA, USA.

Techniques: Isolation, Control, Staining, Flow Cytometry, Injection

Journal: Stem cells and development

Article Title: Discrimination between lung homeostatic and injury-induced epithelial progenitor subsets by cell-density properties.

doi: 10.1089/scd.2012.0468

Figure Lengend Snippet: FIG. 2. Density distribution of common lung cell lineages. (A) Fractionated cells from sham or 2-day bleomycin-treated mice were subjected to flow cytometry and average percent of cells ( – SEM) per fraction are presented. P-value represen- tations shown: normal font—differences in the proportion of protein-expressing cells isolated between fractions from the homeostatic lung; bold font—differences in the proportion of protein-expressing cells between fractions postbleomycin treatment, and italics—overall differences in the proportion of protein-expressing cells between pre- and postbleomycin treatment regardless of fraction. No statistically significant differences were observed in the proportion of CD45-, CD31-, and EpCAM-positive cells between the density fractions in the homeostatic lung. In contrast, cells positive for c-KIT and CD49f were more prevalent in the lower fractions, while SCA-1 expressing cells equilibrated largely in the intermediate fractions. At day 2 following bleomycin treatment, only CD45-positive cells showed significant differences among the fractions equili- brating at the highest density (n ‡ 7; P = 0.01). (B) Representative western blots depicting levels of protein expression per fraction (n ‡ 4). The fibroblastic, smooth muscle, a1-actin (ACTA1) protein signal is highest in fractions 3 and 4 (P < 0.01 by densitometry). Levels of the epithelial (pro-) surfactant protein-C (SFTPC) and secretoglobin family 1A member 1 (SCGB1A1) protein bands are statistically significant between the fractions (P < 0.001 for both). Levels of the AT1 epithelial protein, aquaporin-5 (AQP5) are highest in the lighter fractions (P < 0.001). Bands representing levels of the endothelial CD31 protein and b-actin are also shown.

Article Snippet: Flow cytometry and FACS analysis Fractionated cells were washed and labeled with the following antibodies: FITC-conjugated anti-CD45R (Invitrogen), R-PE anti- SCA-1 (Ly6A/E; Invitrogen), PE/Cy5 antic-KIT (CD117; eBioscience), PE-anti-CD49f (eBioscience), Alexa-488-anti-EpCAM (CD326; Biolegend, San Diego, CA), biotinylated-CD31 (Santa Cruz Biotech) followed by PE/Cy5 streptavidin (Biolegend), and mouse anti-BRDU (ebioscience) followed by anti-mouse Alexa 488 (Invitrogen).

Techniques: Cytometry, Expressing, Isolation, Western Blot

Journal: Stem cells and development

Article Title: Discrimination between lung homeostatic and injury-induced epithelial progenitor subsets by cell-density properties.

doi: 10.1089/scd.2012.0468

Figure Lengend Snippet: FIG. 3. Density properties of lung progenitor cell lineages. Sham or 2-day postbleomycin-treated mice were injected with BrdU, euthanized, and lungs harvested after 1 h. Cells were then dissociated, colabeled for BrdU and CD45, SCA-1, c-KIT, CD31, EpCAM, or CD49f, and processed for flow cytometry. (A) In untreated mice, BrdU-incorporating cells of FR3, primarily belonged to EpCAM and CD49f cell lineages. In contrast, BrdU-incorporating cells of fraction FR4 were SCA-1-positive (n ‡ 5). (B) Bleomycin treatment diminished the presence of BrdU-incorporating EpCAM and CD49f putative progenitor cells in fraction 3 introducing, instead, proliferating CD45, SCA-1, and c-KIT cell lineages to fraction 5 (n ‡ 4). Data are presented as average – SEM. (C) Re- presentative flow cytometry analysis of CD49f and EpCAM expression demonstrating the gating and percentage of sorted CD49f and EpCAMhi cells from density fractions.

Article Snippet: Flow cytometry and FACS analysis Fractionated cells were washed and labeled with the following antibodies: FITC-conjugated anti-CD45R (Invitrogen), R-PE anti- SCA-1 (Ly6A/E; Invitrogen), PE/Cy5 antic-KIT (CD117; eBioscience), PE-anti-CD49f (eBioscience), Alexa-488-anti-EpCAM (CD326; Biolegend, San Diego, CA), biotinylated-CD31 (Santa Cruz Biotech) followed by PE/Cy5 streptavidin (Biolegend), and mouse anti-BRDU (ebioscience) followed by anti-mouse Alexa 488 (Invitrogen).

Techniques: Injection, Cytometry, Expressing

Journal: The Journal of cell biology

Article Title: Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment.

doi: 10.1083/jcb.201005140

Figure Lengend Snippet: Figure 2. Neutrophil migration requires flotillin micro domains. (a) fMLP (1 µg/mouse) was injected into subcutaneous air pouches. At the times indicated, mice were culled and the air pouch lavaged with PBS. Leukocyte counts were per- formed based on cell morphology after staining with Wright- Giemsa; a total of 200 cells were counted per slide. +/+, wild type; /, flotillin 1 knockout. Results are mean ± SEM (n = 3–7); these data are representative of three individual ex- periments. *, P < 0.05. (b) Cellular infiltration of the air pouch was monitored by flow cytometry using CD11b and Ly6G expression as markers of neutrophils (Ly6GhiCD11b+ve) and monocytes (Ly6GloCD11b+ve), as shown by the blue gating regions. Numbers in blue are the percentages of total cells in each gate. (c) Absolute cell numbers calculated from FACS analysis of infiltrating cells as in panel b. Results are mean ± SEM (n = 3–7); data are representative of three individual experiments. *, P < 0.05.

Article Snippet: D ow nloaded from http://rupress.org/jcb/article-pdf/191/4/771/1564314/jcb_201005140.pdf by guest on 01 January 2025 JCB • VOLUME 191 • NUMBER 4 • 2010 778 779Function of flotillins in neutrophil recruitment • Ludwig et al. Phospho-myosin light chain 2 (Ser19; 3671) and phospho-myosin light chain 2 (Thr18/Ser19; 3674) antibodies were from Cell Signaling Technology.

Techniques: Migration, Injection, Staining, Knock-Out, Flow Cytometry, Expressing

Journal: The Journal of cell biology

Article Title: Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment.

doi: 10.1083/jcb.201005140

Figure Lengend Snippet: Figure 5. Flotillin microdomains are required for uropod formation and activation of myosin IIa. (a) Migration of control and flotillin 1/ neutrophils into Matrigel in a gradient of MIP2. The dashed white line indicates the top of the Matrigel layer. Triangles represent the direction of the MIP2 concentration gradient; vertical bar, 50 µm. Images are maximum intensity projections derived from 20–25 confocal Z-slices. (b) Quantification of neutrophil migration into Matrigel. The proportion of cells entering the Matrigel was derived by quantifying the fluorescence above and below the dashed white line in panel a. Combined data from four separate experiments are shown. (c) Indirect immunofluorescence with antibodies against flotillin 1 and -spectrin to show colocalization in the uropod of fMLP-stimulated neutrophils. Bar, 20 µm. (d) Comparison of uropod formation in control (+/+) and flotillin 1/ neutrophils. Cells were fixed 15 min after stimulation with fMLP and labeled with antibodies against CD44. Arrows indicate structures defined as uropods. Bar, 20 µm.

Article Snippet: D ow nloaded from http://rupress.org/jcb/article-pdf/191/4/771/1564314/jcb_201005140.pdf by guest on 01 January 2025 JCB • VOLUME 191 • NUMBER 4 • 2010 778 779Function of flotillins in neutrophil recruitment • Ludwig et al. Phospho-myosin light chain 2 (Ser19; 3671) and phospho-myosin light chain 2 (Thr18/Ser19; 3674) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, Migration, Control, Concentration Assay, Derivative Assay, Fluorescence, Immunofluorescence, Comparison, Labeling

Journal: Nature immunology

Article Title: Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

doi: 10.1038/ni.1645

Figure Lengend Snippet: In vivo administration of rapamycin inhibits YF-17D-induced production of IFN-α in the serum. (a) ELISA of IFN-α production by pDCs from C57BL/6 mice treated daily for 3 d with soluble rapamycin (1.5 mg per kg body weight per day) or rapamycin encapsulated in PLGA microparticles (2 mg per mouse per day), then vaccinated subcutaneously with YF-17D on day 4; blood and spleens were obtained at various times after injection for analysis of serum (right) and pDCs enriched from spleens with microbeads coated with anti–mouse PDCA1 (left). *P < 0.05; **P < 0.001; ***P < 0.0001. Data are representative of at least three independent experiments (error bars, s.d.). (b) Flow cytometry of ovalbumin (OVA) in cells from C57BL/6 mice left untreated (top row) or subcutaneously injected with Alexa Fluor 488–labeled ovalbumin encapsulated in PLGA microparticles (bottom row); draining lymph nodes were isolated 24 h later and treated for 30 min at 37 °C with collagenase type IV, then isolated cells were stained for cell surface lineage markers to identify cell type (above plots). Numbers below outlined areas indicate percent cells containing OVA. cDCs, conventional DCs. Data are representative of three independent experiments.

Article Snippet: For the preparation of splenic pDCs, spleens were digested with collagenase type IV (Worthington Biomedical), and samples were enriched for CD45RA + CD11c int pDCs by the use of microbeads coated with anti–mouse PDCA1 (Miltenyi Biotec).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Flow Cytometry, Labeling, Isolation, Staining

Journal: PLoS ONE

Article Title: Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

doi: 10.1371/journal.pone.0085702

Figure Lengend Snippet: A. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous MCF-7 breast cancer xenografts treated with thiaminase 2000 units SC QOD or buffer control. The median time to endpoint was 41 days for untreated control and 59 days (p = 0.03 Log rank test). B. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous RS4 leukemia subcutaneous xenografts treated with thiaminase 850 units SC BIW or buffer control. The median time to endpoint was 16.5 days for the control group and not reached after 60 days of observation in the treated group (p<0.001 Log rank test). C. Primary ALL and AML specimens were thawed and plated in triplicate in two concentrations of thiaminase 0.4 units/ml and 4 units/ml, and assessed for viability at 48 hours relative to untreated cells. The ALL specimen with the asterisk was used for the in vivo study shown in . D. Primary ALL cells were injected IV on Day 1; three thiaminase treatments of 2000 units/kg SC were administered on days 17, 20 and 24. The animals were sacrificed on Day 33 and bone marrow was examined by flow cytometry for human ALL cells (percent human CD45); Untreated n = 4; vehicle treated n = 10, native thiaminase n = 8 (* p<0.01, Mann-Whitney test).

Article Snippet: The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile.

Techniques: Control, In Vivo, Injection, Flow Cytometry, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

doi: 10.1371/journal.pone.0085702

Figure Lengend Snippet: A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium, thiamine-free medium or medium containing thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control, N3PT alone, 1k-PEGylated thiaminase and 1k-PEGylated thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control, 23 days for N3PT, 25 days for 1k-PEG thiaminase and 55 days for 1k-PEG thiaminase (p<0.01 Log rank test). D. Oxygen consumption rate of RS4 cells treated with thiaminase, N3PT or thiaminase followed by N3PT (clear: control; thin-stripe: thiaminase; thick- stripe: N3PT; solid: thiaminase + N3PT).

Article Snippet: The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile.

Techniques: Inhibition, Incubation, Control

Journal: PLoS ONE

Article Title: Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

doi: 10.1371/journal.pone.0085702

Figure Lengend Snippet: Both cell lines were analyzed under six conditions: control for 24 hours (C-24); incubation in thiaminase for 24 hours (T-24); control for 48 hours (C-48); thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle, the rectangle dimensions reflect the range of the two mid-quartile values, and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p<0.05 and * indicates 0.05

Article Snippet: The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile.

Techniques: Control, Incubation, Biomarker Discovery, Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

doi: 10.1371/journal.pone.0085702

Figure Lengend Snippet: A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after thiaminase treatment- most notably isovalerylcarnitine, which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT), the enzymes that catalyzes the reactions that produce BCKDH substrates, and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1, respectively) in RS4 leukemia cells treated with thiaminase, rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p<0.05 and * indicates 0.05

Article Snippet: The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile.

Techniques: Inhibition, Western Blot, Cytotoxicity Assay, Control

Journal: PLoS ONE

Article Title: Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

doi: 10.1371/journal.pone.0085702

Figure Lengend Snippet: A. Accumulation of the products of phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (hydroxyphenylpyruvate and hydroxyphenyllactate) catabolism in RS4 cells after treatment with thiaminase for 48 hours. B. Accumulation of tryptophan catabolites after thiaminase treatment, showing accumulation of indolelactate but not kynurenine. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p<0.05 and * indicates 0.05

Article Snippet: The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile.

Techniques:

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: (A) Phenotypic characterization of BMDMs by flow cytometry. Mouse BMDMs were analyzed by flow cytometry for CD11b and F4/80 cell surface markers to identify the differentiation of bone marrow cells into macrophages. Over 95% of cells within cultures are double positive. (B) Dose- and time- dependant effect of LPS on SIRT1 protein expression. After BMDMs were incubated with various doses of LPS for 24 h (upper panel) and 100 ng/ml LPS for the indicated times (lower panel), western blot were performed with β-actin as a loading control. (C) Time-dependant effect of LPS on SIRT1 mRNA expression. After BMDMs were incubated with 100 ng/ml LPS for the indicated times, real-time PCR were performed. (D) The effects of signal transduction inhibitors on LPS-induced SIRT1 expression. BMDMs were pretreated with 10 μM PD098059 (PD), 10 μM SB203580 (SB), 10 μM SP600125 (SP), 10 μM PDTC, 1 μM wortmannin (WT), 5 μM LY294002 (LY), 5 μM JAK1 inhibitor (JAK1), and 10 μM JAK3 inhibitor (JAK3), for 1 h and further treated with LPS for 24 h. Full-length blots are presented in . The Western blot shown is a representative of three independent experiments.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Flow Cytometry, Expressing, Incubation, Western Blot, Control, Real-time Polymerase Chain Reaction, Transduction

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: (A) Dose dependant effect of IFN-β on SIRT1 protein expression. BMDMs were incubated with the indicated doses of IFN-β for 24 h. A western blot was performed for SIRT1 with β-actin as a loading control. (B) The effect of IFN-β neutralizing antibody on LPS-induced SIRT1 expression. BMDMs were treated with LPS in the presence or absence of IFN-β neutralizing antibody (1 μg/mL) for 24 h. (C) The effect of various inhibitors for signal transduction on IFN-β-induced SIRT1 expression. BMDMs were pretreated with vehicle (DMSO), 10 μM PD098059 (PD), 10 μM SB203580 (SB), 10 μM SP600125 (SP), 10 μM PDTC, 1 μM wortmannin (WT), 5 μM LY294002 (LY), 5 μM JAK1 inhibitor (JAK1), and 10 μM JAK3 inhibitor (JAK3) for 1 h and further treated with 100 U/ml IFN-β for 24 h. Full-length blots are presented in . The Western blot shown is a representative of three independent experiments.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Control, Transduction

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: (A) The effect of SIRT1 expression by adenovirus-SIRT1 on the LPS-induced release of inflammatory cytokines. BMDMs were infected with adenovirus-SIRT1 (10,000 MOI) for 24 h, and then incubated with new media supplemented with LPS (100 ng/mL) for 24 h. Western blotting was performed for SIRT1 using α-tubulin as a loading control (left panel). The levels of several inflammatory cytokines in the cell culture supernatants were measured by ELISA (right panel). (B) The effect of IFN-β treatment on LPS-induced inflammatory cytokine release. BMDMs were pretreated with 100 U/mL IFN-β for 24 h, and then supplemented with LPS (100 ng/mL) for 24 h. Western blotting was performed for SIRT1 using α-tubulin as a loading control (left panel). The levels of several inflammatory cytokines in cell culture supernatants were measured by ELISA (right panel). The results are represented as means ± SEM, * P < 0.05, and ** P < 0.01. Full-length blots are presented in . The Western blot shown is a representative of three independent experiments.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Incubation, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: (A) Survival rate of mice infected with SIRT1-expressing adenovirus in response to LPS challenge or cecal ligation puncture. C57BL/6 mice were randomly grouped and injected with LacZ or SIRT1 expressing adenovirus (1 × 10 10 MOI). Two days later, the mice were injected with 18 mg/kg body weight of LPS, and ligation and puncture of the cecum (CLP) or sham operation was performed. SIRT1 expression in the liver and spleen of mice infected with SIRT1 expressing adenovirus was checked by western blot (upper panel). The survival rates at different times after LPS challenge (middle panel) or CLP (lower panel) are shown. (B) Survival rate of mice treated with IFN-β in response to LPS challenge or cecal ligation puncture. C57BL/6 mice were injected with vehicle or IFN-β (1000 U/20 g). SIRT1 expression in the liver and spleen of mice treated with IFN-βwas checked by western blot (upper panel). Thirty min later, the mice were injected with 18 mg/kg body weight of LPS, and ligation and puncture of the cecum or sham operation was performed. The survival rates at different times after LPS challenge (middle) or CLP (lower) are shown. Full-length blots are presented in . The Western blot shown is a representative of three independent experiments.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Ligation, Injection, Western Blot

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: Morphological changes in mouse kidney (A) and spleen (B) sections infected with LacZ- or SIRT1-expressing adenovirus for 48 h, or treated with vehicle or IFN-β for 30 min before LPS challenge (magnification, ×40). Organ sections were obtained from mice 15 h after treatment with LPS at 18 mg/kg body weight, perfused and fixed with formalin, and stained with hematoxylin and eosin.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Staining

Journal: Scientific Reports

Article Title: Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

doi: 10.1038/srep04220

Figure Lengend Snippet: C57BL/6 mice were randomly grouped and injected with DN SIRT1 expressing adenovirus (1 × 10 10 MOI). Two days later, the mice were injected with vehicle or IFN-β (1000 U/20 g) and 30 min later further injected with 18 mg/kg body weight of LPS. Full-length blots are presented in . The Western blot shown is a representative of three independent experiments.

Article Snippet: Anti-mouse SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Injection, Expressing, Western Blot