Journal: Gut microbes

Article Title: Host miRNA-21 promotes liver dysfunction by targeting small intestinal Lactobacillus in mice.

doi: 10.1080/19490976.2020.1840766

Figure Lengend Snippet: Figure 1. miR-21KO mice are protected from bile duct ligation (BDL)-induced liver injury. (a) Representative images of hematoxylin and eosin (H&E) (upper panel) and TUNEL (lower panel) stained liver sections after sham operation and in WT and miR-21KO mice 3 days after BDL. Apoptotic cells are shown in red and nuclei are counterstained in blue with Hoechst 33258 dye. Scale bar, 50 μm. (b) Histology scores of inflammation and necrosis, and quantification of TUNEL-positive cells/mm2 in WT and miR-21KO mice 3 days after BDL. (c) Serum alkaline phosphatase (AP), alanine aminotransferase (ALT) and total bile acids in WT and miR-21KO mice after either sham operation or BDL for 3 days. (d) liver mRNA expression of Col1α1, α-Sma and Tgf-β in WT and miR-21KO mice after either sham operation or BDL for 3 days. (e) liver hydroxyproline levels and α-SMA protein in WT and miR-21KO mice after either sham operation or BDL for 3 days. Results are expressed in fold change as mean values with error bars ± SEM of 4–6 individual mice. Data were statistically analyzed with ANOVA Tukey’s multiple comparisons test *p < .05; **p < .01 and ***p < .001.

Article Snippet: Serum was collected for the evaluation of alanine aminotransferase (ALT) and alkaline phosphatase (AP) (ABX ALT and AP assay kit; Horiba) and total bile acids (3αhydroxysteroid dehydrogenase enzymatic assay kit; Randox Reagents).

Techniques: Ligation, TUNEL Assay, Staining, Expressing

Journal: Gut microbes

Article Title: Host miRNA-21 promotes liver dysfunction by targeting small intestinal Lactobacillus in mice.

doi: 10.1080/19490976.2020.1840766

Figure Lengend Snippet: Figure 5. Lactobacillus reuteri supplementation protects from bile duct ligation (BDL)-induced liver damage. (a) Representative images of hematoxylin and eosin (H&E) stained liver sections from control and Lactobacillus reuteri DSM 17938 (L. reuteri) supplemented mice after BDL for 3 days. (b) Histology scores for hepatocyte hypertrophy, lipidosis, bile duct hyperplasia, necrosis and inflammation in control and L. reuteri supplemented mice 3 days after BDL. (c) Serum alkaline phosphatase (AP), alanine aminotransferase (ALT) and total bile acids in control and L. reuteri supplemented mice 3 days after BDL. (d) mRNA expression of liver fibrosis markers Col1α1, α-Sma and Tgf-β in control and L. reuteri supplemented mice after BDL for 3 days. (e) Liver hydroxyproline and α-SMA protein levels in control and L. reuteri supplemented mice after BDL for 3 days. (f) mRNA expression levels of liver inflammatory markers Tnf-α, Il-1β, Mip-2 and Tlr-4 in control and L. reuteri supplemented mice after BDL for 3 days. (g) qPCR mRNA expression levels of L. reuteri in small intestinal lumen samples of control and L. reuteri supplemented mice after BDL for 3 days. (h) qPCR mRNA expression levels of liver D-Lactate dehydrogenase (D-Ldh) in control and L. reuteri supplemented mice after BDL for 3 days. (i) mRNA expression and protein levels of TGF-β in mouse macrophages stimulated with 1 mM of either D-Lactate or L-Lactate. Mean values of the in vivo experiments were calculated as fold change versus control with error bars ± SEM of 4–6 individual mice. Statistical analysis performed using unpaired t-test. Mean values of the in vitro assays were calculated as fold change versus control macrophages with error bars ± SEM of 5 individual experiments. Statistical analysis was performed with ANOVA Tukey’s multiple comparisons test. *p < .05, **p < .01 and ***p < .001.

Article Snippet: Serum was collected for the evaluation of alanine aminotransferase (ALT) and alkaline phosphatase (AP) (ABX ALT and AP assay kit; Horiba) and total bile acids (3αhydroxysteroid dehydrogenase enzymatic assay kit; Randox Reagents).

Techniques: Ligation, Staining, Control, Expressing, In Vivo, In Vitro

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control

Journal: Cell death & disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: Fig. 5 AP2ε regulates phenotypic plasticity of melanoma cells. A Representative Western Blot images depicting murine MITF and BRN2 protein levels in primary AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (left panels) and human MITF and BRN2 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (right panels). β-Actin served as loading control. B Immunohistochemical staining of MITF and BRN2 protein expression in Mel Im melanoma cells bioprinted in CIB (Magnification: 20x). Quantification of percentage of cells with “no”, “weak”, or “strong” MITF and BRN2 expression. Data are represented as mean ± SEM; *p < 0.05; ns: not significant (Two-way- ANOVA with Fisher’s LSD test). C mRNA- and protein expression analysis for Snail1 in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). D mRNA- and protein expression analysis for Snail1 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). E mRNA- and protein expression analysis for E-cadherin in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). F mRNA- and protein expression analysis for E-cadherin in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). Data information: All data from at least three independent experiments are represented as mean ± SEM; *p < 0.05 (Two-tailed Student’s t-test; ns: not significant).

Article Snippet: Primary antibodies against MITF (1:500 in 5% BSA, Santa Cruz, sc-515925, RRID:AB_2828036), BRN2 (1:500 in 5% BSA/1x TBS-T, Santa Cruz, sc393324, RRID:AB_2737347), Snail1 (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3879, RRID:AB_2255011) and E-cadherin (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3195, RRID:AB_2291471) were incubated overnight at 4 °C.

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Control, Immunohistochemical staining, Staining, Expressing, Two Tailed Test

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay