Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: Cadherin-20 (CDH20) , R & D Systems , 5604-CA , ECM.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: ( A ) Process of formation of successive biosensor layers. Curves I–V presents five different CDH12 concentrations; ( B ) Langmuir curve.

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: Intra-run and inter-run precision and accuracy.

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: Influence of potential interferents on CDH12 assays.

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: Passing–Bablok regression curves comparing the results of the SPRi CDH12 and ELISA methods performed on ( A ) plasma samples and ( B ) peritoneal fluid samples. The blue solid line marks the Passing–Bablok regression curve; the black fine dashed line indicates where the bias would be zero (SPRi CDH12 = ELISA).

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: WHO’s ASSURED criteria for SPRi CDH12 tests.

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques: Clinical Proteomics, Diagnostic Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

doi: 10.3390/ijms242316894

Figure Lengend Snippet: Schematic representation of the procedures for biosensor preparation, CDH12 quantitative analysis, and biosensor regeneration.

Article Snippet: Monoclonal recombinant rat anti-CDH12 antibody (Cat. No. MAB2240) and recombinant human CDH12 protein (Cat. No. 2240-CA), each >95% pure, were purchased from R&D Systems.

Techniques:

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: Cadherin-8 (CDH8) , R & D Systems , 188-C8 , ECM.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: Scientific reports

Article Title: PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer.

doi: 10.1038/s41598-024-59427-3

Figure Lengend Snippet: Figure 6. PAFAH1B3 affects the expression of EMT-related proteins in pancreatic cancer cells (A) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. (B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing sh-PAFAH1B3 or sh-NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. β-Actin was used as an internal control. The data represent the average of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: rabbit anti-PAFAH1B3 polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), mouse anti-β-actin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-PCNA polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Ncadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Snail1 polyclonal antibody 3 Vol.

Techniques: Expressing, Transduction, Western Blot, Control

Journal: Science immunology

Article Title: Persistent CAD activity in memory CD8 + T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge

doi: 10.1126/sciimmunol.abh4271

Figure Lengend Snippet: (A) The de novo pyrimidine synthesis pathway. The rate-limiting trifunctional enzyme CAD is phosphorylated and activated by S6K1, which is in turn phosphorylated and activated by mTORC1. The CAD protein catalyzes the first three steps of this pathway and is inhibited by the small molecule PALA (shown in red). (B) Western blot analysis of CD8+ T cells isolated from spleens of naïve WT mice and stimulated with anti-CD3/28 antibodies for indicated time periods with or without rapamycin (100 nM). (C) Western blot analysis of CD8+ T cells isolated from spleens of naïve WT, Rptor−/− and Rictor−/− mice stimulated with anti-CD3/28 antibodies for indicated three hours. (D) Flow cytometric analysis of WT, Rptor−/− and Rictor−/− mice for pCAD. CD8+ T cells from WT mice were analyzed as either unstimulated (grey) or stimulated using anti-CD3/28 antibodies for three hours with 100 nM rapamycin (black) or without rapamycin (red). Rptor−/− (blue) and Rictor−/− (green) CD8+ T cells were also stimulated for three hours. (E) Western blot analysis of CD8+ T cells isolated from spleens of naïve WT mice and stimulated with anti-CD3/28 antibodies for 36 and 72 hours. (F) Western blot analysis of CD8+ T cells isolated from spleens of naïve C57BL/6J mice and stimulated with anti-CD3/28 antibodies for 48 hours, then expanded in media containing IL-2 or IL-7 + IL-15 for an additional five days. Data in (B) includes representative image and densitometry of fold increase in signal relative to actin across three experiments. Data in (C) includes representative image and densitometry of signal relative to actin across three experiments. Data in (D) included representative image and depicts fold change in MFI over naïve CD8+cells across three experiments. Data in (E) and (F) include representative image and densitometry of signal ratio of phosphoprotein to total protein relative to actin across three experiments. Data in (B), (E), and (F) analyzed by two-tailed t test. Data in (C) and (D) analyzed by one-way ANOVA followed by Tukey’s HSD test. *P <0.05, ns = not significant.

Article Snippet: Primary antibodies included CD8α (53–6.7), CD44 (IM7), IL-2 (JES6-5H4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), CD45RO (UCHL1), CD45RA (HI100), CD3 (UCHT1), IFN-γ (B27), and TNF-α ((RUO(GMP)) from BD Biosciences, CD62L (MEL-14) from BioLegend, anti-CAD (11933S), anti pCAD (S1859, 12662S), anti-S6 (2217), anti-pS6 (S240/244, D68F8), anti S6K (9202), anti-pS6K (T389, 9205S), anti-RPL5 (14568S), and anti-β-Actin (D6A8, 8457) from Cell Signaling, Fixable Viability Dye eFluor 780 (65-0865-14) from eBioscience, anti-HA Alexa Fluor 488 conjugated (F-7, sc-7392) from Santa Cruz Biotechnology, and anti-RPL22 (PA5-97192) from ThermoFisher.

Techniques: Western Blot, Isolation, Two Tailed Test

Journal: Science immunology

Article Title: Persistent CAD activity in memory CD8 + T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge

doi: 10.1126/sciimmunol.abh4271

Figure Lengend Snippet: (A) Flow cytometric analysis of pCAD signal in naïve cells (CD8+CD44−CD62L+) and CM cells (CD8+CD44+CD62L+) isolated from the spleens of uninfected WT mice. (B) Flow cytometric analysis of pCAD in naïve and antigen-specific CM CD8+ cells. CD8+ cells were isolated from spleens of naïve OT-I mice and adoptively transferred into WT hosts which were immediately infected with Vac-OVA with splenocytes harvested forty five days later. (C) Flow cytometric analysis of pCAD and pS6 during viral infection. Experiment was performed as in (B) with splenocytes harvested eight days and fifteen days post-infection. (D) Schematic of in vivo rapamycin administration. Mice were administered Vehicle, Low Dose (75 μg/kg) or High Dose (3 mg/kg) rapamycin for Day −1 to Day 8 of Listeria-OVA infection with adoptive transfer of cells freshly isolated from naïve OT-I mice on Day 0. Splenocytes were harvested on Day 35 of infection for analysis by flow cytometry. (E) Summary pCAD intensity and Thy1.1+ data from splenocytes in experiments described in (D) across three replicates. Data in (A) and (B) depict representative gating and fluorescent staining alongside summary MFI values ± SD across three independent experiments normalized to naïve CD8+ cell values within each experiment analyzed by paired two-tailed t-test (n=5 mice per experiment). Data in (C) show mean fluorescence intensity values across three independent experiments normalized to naïve values within each experiment analyzed by paired two-tailed t-test (n=3 mice per experiment). Data in (E) depict representative gating and percentage of Thy1.1+ cells among CD8+ cells and pCAD MFI values ± SD across three independent experiments normalized to vehicle treated CD8+ cell values within each experiment analyzed by one-way ANOVA followed by Tukey’s HSD test. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns = not significant.

Article Snippet: Primary antibodies included CD8α (53–6.7), CD44 (IM7), IL-2 (JES6-5H4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), CD45RO (UCHL1), CD45RA (HI100), CD3 (UCHT1), IFN-γ (B27), and TNF-α ((RUO(GMP)) from BD Biosciences, CD62L (MEL-14) from BioLegend, anti-CAD (11933S), anti pCAD (S1859, 12662S), anti-S6 (2217), anti-pS6 (S240/244, D68F8), anti S6K (9202), anti-pS6K (T389, 9205S), anti-RPL5 (14568S), and anti-β-Actin (D6A8, 8457) from Cell Signaling, Fixable Viability Dye eFluor 780 (65-0865-14) from eBioscience, anti-HA Alexa Fluor 488 conjugated (F-7, sc-7392) from Santa Cruz Biotechnology, and anti-RPL22 (PA5-97192) from ThermoFisher.

Techniques: Isolation, Infection, In Vivo, Adoptive Transfer Assay, Flow Cytometry, Staining, Two Tailed Test, Fluorescence

Journal: Science immunology

Article Title: Persistent CAD activity in memory CD8 + T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge

doi: 10.1126/sciimmunol.abh4271

Figure Lengend Snippet: (A-C) CD8+ cells isolated from WT mice were stimulated with anti-CD3/28 antibodies for 24 hours before introduction of indicated retroviral constructs with cells analyzed on Day 6 of culture. (A) Western blot analysis of pCAD (S1859) and total CAD levels in CD8+ cells following retroviral transduction with EV-MigR1 or CAD-MigR1 constructs. (B) Percentage M+1 peak data for de novo pyrimidine synthesis metabolites measured by LC/MS between EV-MigR1 and CAD-MigR1 cells. On Day 6 of culture, freshly-isolated naïve, EV-MigR1 and CAD-MigR1-transduced cells were rested in culture media before transfer to glutamine-free media supplemented with 4 mM amide-labeled 15-N glutamine, with a portion of each condition stimulated with anti-CD3/28 antibodies. (C) Summary data of cytokine response of retrovirally-transduced cells across three experiments generated as in (B) following 4 hours stimulation with PMA/ionomycin. Data gated on live CD8+GFP+ cells with percentage IFNγ+TNFα+ cells reported. (D) Schematic of adoptive transfer experiments with previously activated P14 retrovirally-transduced cells. (E) Percentage GFP+ before transfer and at Day 4 and Day 6 following adoptive transfer and infection with LCMV-Armstrong. Data in (A) depict representative western blot images and densitometry quantification of indicated protein relative to actin from three independent experiments analyzed by two-tailed t test. Data in (B) are mean ± SD of triplicate samples representative of two independent experiments analyzed by two-way ANOVA followed by Tukey’s HSD test. Data in (C) depicts percentage double positive cells across three independent experiments analyzed by two-tailed t test. Data in (E) depict representative gating strategies and GFP+ ± SD analyzed with two-tailed t test at each indicated time point (n=5 mice per condition). *P <0.05, **P<0.01, ***P<0.001.

Article Snippet: Primary antibodies included CD8α (53–6.7), CD44 (IM7), IL-2 (JES6-5H4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), CD45RO (UCHL1), CD45RA (HI100), CD3 (UCHT1), IFN-γ (B27), and TNF-α ((RUO(GMP)) from BD Biosciences, CD62L (MEL-14) from BioLegend, anti-CAD (11933S), anti pCAD (S1859, 12662S), anti-S6 (2217), anti-pS6 (S240/244, D68F8), anti S6K (9202), anti-pS6K (T389, 9205S), anti-RPL5 (14568S), and anti-β-Actin (D6A8, 8457) from Cell Signaling, Fixable Viability Dye eFluor 780 (65-0865-14) from eBioscience, anti-HA Alexa Fluor 488 conjugated (F-7, sc-7392) from Santa Cruz Biotechnology, and anti-RPL22 (PA5-97192) from ThermoFisher.

Techniques: Isolation, Retroviral, Construct, Western Blot, Transduction, Liquid Chromatography with Mass Spectroscopy, Labeling, Generated, Adoptive Transfer Assay, Infection, Two Tailed Test

Journal: Science immunology

Article Title: Persistent CAD activity in memory CD8 + T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge

doi: 10.1126/sciimmunol.abh4271

Figure Lengend Snippet: (A) Flow cytometric analysis of pCAD in naïve (CD45RA+CD45RO−) and central memory (CD45RA−CD45RO+CCR7+) human CD3+CD8+ cells. PBMCs were freshly isolated from healthy donor blood before fixation and analysis. (B) Flow cytometric analysis of pCAD levels in Cad-overexpressing (pcDNA3-CAD) or empty vector-expressing human (pcDNA3-EV) cells. Data shown are gated on live CD3+CD8+HA+ cells. (C) Schematic for overexpression of HA-tagged CAD construct in cultured human CD8+ T cells. (D) Flow cytometric analysis of IFNγ and TNFα production by CD8+ cells with and without Cad overexpression following indicated drug treatment. Cells were exposed to drug for 24 hours before drug removal and four hours of restimulation with PMA/ionomycin. Data shown are gated on CD3+CD8+HA+ cells. (E) Western blot of ribosomal proteins in naïve and memory CD8+ T cells. Human PBMCs were thawed and magnetically sorted into naive (CD8+CD45RO−) and memory (CD8+CD45RO+) populations for lysis and analysis. Data in (A) representative of three healthy donors and is depicted alongside representative gating. Data in (B), (D), and (E) depict representative images as well as summary data across three independent experiments with three frozen PBMC aliquots from three separate healthy donors with data in (B) and (E) analyzed by two-tailed t test and data in (D) analyzed by two-way ANOVA followed by Tukey’s HSD test. *P <0.05, **P<0.01, ***P<0.001.

Article Snippet: Primary antibodies included CD8α (53–6.7), CD44 (IM7), IL-2 (JES6-5H4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), CD45RO (UCHL1), CD45RA (HI100), CD3 (UCHT1), IFN-γ (B27), and TNF-α ((RUO(GMP)) from BD Biosciences, CD62L (MEL-14) from BioLegend, anti-CAD (11933S), anti pCAD (S1859, 12662S), anti-S6 (2217), anti-pS6 (S240/244, D68F8), anti S6K (9202), anti-pS6K (T389, 9205S), anti-RPL5 (14568S), and anti-β-Actin (D6A8, 8457) from Cell Signaling, Fixable Viability Dye eFluor 780 (65-0865-14) from eBioscience, anti-HA Alexa Fluor 488 conjugated (F-7, sc-7392) from Santa Cruz Biotechnology, and anti-RPL22 (PA5-97192) from ThermoFisher.

Techniques: Isolation, Plasmid Preparation, Expressing, Over Expression, Construct, Cell Culture, Western Blot, Lysis, Two Tailed Test