Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Collagen synthesis. ( A ) Basal collagen synthesis. ( B ) Collagen synthesis after TGF-β (10 nM) stimulation for 24 h. Measurement based on incorporation of [ 3 H]-proline into collagen. Data are mean ± SEM of triplicate determinations. ( C ) Cells treated with or without TGF-β for 24 h were lysed and proteins subjected to immunoblotting using antibodies against TGF-β receptor I and II, phospho-Smad-2, Smad-2, phospho-Smad-3, and Smad-3. GAPDH was used as loading control. * p < 0.05, ** p < 0.01 comparing average of hPSCs with HPaSteC, i-mPSCs for ( A ); ** p < 0.01 comparing control (non-treated) cells with TGF-β treated cells for ( B ). PSC, pancreatic stellate cell; hPSC, human primary PDAC-derived PSC culture; HPaSteC, PSCs from normal human pancreas; i-hPSC, immortalized human PSCs; i-mPSC C2 and C3, immortalized mouse PSCs clone 2 and 3.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Western Blot, Derivative Assay

Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Characteristics of the various pancreatic stellate cell (PSC) cultures.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Isolation, Gradient Centrifugation, Transfection, Expressing, DNA Synthesis, Migration

Journal: Cell

Article Title: Parental genome unification is highly error-prone in mammalian embryos

doi: 10.1016/j.cell.2021.04.013

Figure Lengend Snippet:

Article Snippet: As primary antibodies we used human anti-centromere antibody (ACA) at 1:250 dilution (Antibodies Incorporated #15-234), rat anti-Nup98 at 1:50 (Abcam #ab50610), mouse anti-NPC/MAb414 at 1:100 (Covance #MMS-120P), rat anti-α-tubulin at 1:1000 (AbD Serotec #MCA78G), rabbit anti-β-8-tubulin at 1:500 (Sigma-Aldrich #SAB2700070), mouse anti-Trf1 at 1:250 (Alpha diagnostic international #TRF12-S), mouse anti-Histone at 1:100 (Merck #MAB3422), mouse anti-γ-tubulin at 1:250 (Sigma-Aldrich #T5326), rabbit anti-Sun1 at 1:100 (CST #8886), rabbit anti-Lamin B1 at 1:100 (Abcam #ab16048) and mouse anti-Lamin A/C at 1:50 (Sigma-Aldrich #MABT1340), rabbit anti-TPR at 1:100 (Abcam #ab84516), rabbit anti-Elys at 1:100 (Novus biological #NBP1-87952), mouse anti-Nup153 at 1:100 (Abcam #ab96462).

Techniques: Recombinant, RNA HS Assay, Cloning, cDNA Synthesis, Plasmid Preparation, Software, Microscopy

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay

Journal:

Article Title: Tolerance induced by inhaled antigen involves CD4 + T cells expressing membrane-bound TGF-? and FOXP3

doi: 10.1172/JCI200420509

Figure Lengend Snippet: Cell contact and TGF-β–dependent inhibition of proliferation by CD4+ T cells from tolerized mice. (A) Mice were first exposed to PBS (inflammation group) or 1% OVA (tolerance group) daily for 10 days and then were immunized with OVA/alum on days 21 and 27. Splenic CD4+ T cells isolated on day 34 were stimulated in vitro with different concentrations of OVA (10–200 ∝g/ml) and APCs at equivalent cell numbers (105 cells per well). Cells were mixed as described in the legend to Figure ​Figure22 or separated by transwell. In the transwell experiments, cells from the inflammation group were plated in the wells, and cells from tolerized mice on the insert and thymidine incorporation in the former group was measured. *P < 0.05 versus proliferation of cells in the inflammation group. (B) Chicken IgY anti–TGF-β1 (100 ng/ml) or isotype control (chicken IgY) was added to mixed cultures. **P < 0.05 of mixed cultures incubated with anti–TGF-β1 compared with mixed cultures incubated without Ab. (C) Anti–IL-10 (1 mg/ml) or isotype control was added to mixed cultures. All assays were incubated for 72 hours, after which the cells were pulsed for measurement of [3H]-thymidine incorporation. Each data point represents the mean plus or minus SEM of triplicate wells. Shown is a representative experiment of three experiments.

Article Snippet: In some experiments, neutralizing Ab or relevant, matching isotype controls were added to the mixture of CD4 + T cells from the inflammation and tolerance groups as follows: anti–TGF-β1 at 50 ng/ml and 100 ng/ml (R&D Systems Inc.); isotype control: normal chicken IgY (R&D Systems Inc.) at 100 ng/ml (shown); monoclonal anti-mouse IL-10 Ab (R&D Systems Inc.) at 0.1 ∝g/ml and 1 ∝g/ml (shown); and rat IgG1 isotype control (R&D Systems Inc.) at 1 ∝g/ml.

Techniques: Inhibition, Isolation, In Vitro, Incubation

Journal:

Article Title: Tolerance induced by inhaled antigen involves CD4 + T cells expressing membrane-bound TGF-? and FOXP3

doi: 10.1172/JCI200420509

Figure Lengend Snippet: Expression of membrane-bound TGF-β on freshly isolated cells from both groups of mice and their similar cytokine secretion profile. (A) Cell surface TGF-β and CD25 expression on freshly isolated CD4+ T cells from inflammation and tolerance groups. The boxed area denotes cells expressing high levels of CD25. (B) CD4+ T cells from the two groups’ cells were cultured with OVA/APCs for two rounds of stimulation (maintaining equal numbers of cells during restimulation). Cells expressing cell surface TGF-β were isolated using PE-labeled anti–TGF-β Ab, anti-PE microbeads, and separation on magnetic columns. Equal numbers of positively selected cells were restimulated with OVA/APCs for 72 hours, and the indicated cytokines in the culture supernatants were measured by ELISA. Cells expressing IL-10 were isolated using the MACS IL-10 secretion assay, stimulated, and assessed for cytokine production as described above. All data are representative of two independent experiments.

Article Snippet: In some experiments, neutralizing Ab or relevant, matching isotype controls were added to the mixture of CD4 + T cells from the inflammation and tolerance groups as follows: anti–TGF-β1 at 50 ng/ml and 100 ng/ml (R&D Systems Inc.); isotype control: normal chicken IgY (R&D Systems Inc.) at 100 ng/ml (shown); monoclonal anti-mouse IL-10 Ab (R&D Systems Inc.) at 0.1 ∝g/ml and 1 ∝g/ml (shown); and rat IgG1 isotype control (R&D Systems Inc.) at 1 ∝g/ml.

Techniques: Expressing, Membrane, Isolation, Cell Culture, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Cell Stem Cell

Article Title: CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion

doi: 10.1016/j.stem.2020.10.015

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CS , Novus Biologicals , NBP2-13878.

Techniques: Virus, Recombinant, Isolation, Bradford Protein Assay, cDNA Synthesis, SYBR Green Assay, DC Protein Assay, Software

Journal: The Journal of reproduction and development

Article Title: Expression pattern of progranulin in the human placenta and its effect on cell proliferation in the choriocarcinoma cell line BeWo.

doi: 10.1262/jrd.10-073k

Figure Lengend Snippet: Fig. 1. A: Immunoblot detection of progranulin protein in human placenta lysates at different times of gestation. SDS-PAGE (10% acrylamide) was performed with 10 μg protein per slot. Recombinant human progranulin (1 μg) was used as a positive control (left lane). The two bands represent the glycosylated (88 kDa) and deglycosylated (68 kDa) protein. B: Quantified staining intensity of Fig. 1A. Values represent the amount of both bands per lane (i.e., the glycosylated and deglycosylated forms). C: Semiquantitative measurement of the staining intensity of the villous trophoblast cells in representative immunostained pictures. Values are shown as means + SD. The levels of the 8th week and 39th week specimens are significantly different (Mann-Whitney U test, n=6).

Article Snippet: For quantification of progranulin levels, we used the monoclonal Quantikine® human progranulin immunoassay (R&D Systems) following the instructions provided in the manual.

Techniques: Western Blot, SDS Page, Recombinant, Positive Control, Staining, MANN-WHITNEY

Journal: The Journal of reproduction and development

Article Title: Expression pattern of progranulin in the human placenta and its effect on cell proliferation in the choriocarcinoma cell line BeWo.

doi: 10.1262/jrd.10-073k

Figure Lengend Snippet: Fig. 2. Representative photographs of immunostaining of frozen sections of first and third trimester placentae. A: progranulin, 7th week p.m. B: progranulin, 8th week p.m. C: progranulin, 14th week p.m. D: progranulin, 39th week p.m. E: progranulin (without hemalaun), 12th week p.m. F: negative control without primary antibody (without hemalaun), 8th week p.m. Intensity of staining was strongest during the first trimester and clearly diminished at term (see Fig. 1C). Progranulin was mainly localized to the villous trophoblast cells (arrows), whereas expression in the villous stroma (asterisks) was only weak. Scale bar=100 μm.

Article Snippet: For quantification of progranulin levels, we used the monoclonal Quantikine® human progranulin immunoassay (R&D Systems) following the instructions provided in the manual.

Techniques: Immunostaining, Negative Control, Staining, Expressing

Journal: The Journal of reproduction and development

Article Title: Expression pattern of progranulin in the human placenta and its effect on cell proliferation in the choriocarcinoma cell line BeWo.

doi: 10.1262/jrd.10-073k

Figure Lengend Snippet: Fig. 3. Differential expression of progranulin (upper lane, green) using double immunofluorescence staining. Corresponding frozen sections stained for cytokeratin 7 (lower lane, red). A–C: 12th week p.m. D: 39th week p.m. A: Strong progranulin expression of the glandular endometrial epithelium (arrow) with only weak staining of surrounding decidual stroma (asterisks). B: Maternofetal interface. Placental villi with strong progranulin expression of the villous trophoblast cells (VT, arrow). The cytokeratin 7-positive cells in the decidua on the left side of the photographs correspond to extravillous trophoblast cells (EVT, arrowhead). In contrast to VT, the EVT did not obviously express progranulin. C: Placental villus with an outgrowing cell island of EVT cells (arrowhead). Progranulin expression of EVT is weak like that in B. D: Maternofetal interface (39th week). Cytokeratin 7-positive VT (arrow) and EVT (arrowhead). The progranulin expression in the VT is much weaker compared with first trimester specimens. Scale bar=100 μm.

Article Snippet: For quantification of progranulin levels, we used the monoclonal Quantikine® human progranulin immunoassay (R&D Systems) following the instructions provided in the manual.

Techniques: Quantitative Proteomics, Double Immunofluorescence Staining, Staining, Expressing

Journal: The Journal of reproduction and development

Article Title: Expression pattern of progranulin in the human placenta and its effect on cell proliferation in the choriocarcinoma cell line BeWo.

doi: 10.1262/jrd.10-073k

Figure Lengend Snippet: Fig. 4. Immunostaining of progranulin. A: Isolated trophoblast cells after 72 h of culture (9th week of gestation). About one third of the cells show strong protein expression. B: Negative control without primary antibody. C: Jeg-3 cells, 6th passage. D: BeWo cells, 5th passage. Scale bar=100 μm.

Article Snippet: For quantification of progranulin levels, we used the monoclonal Quantikine® human progranulin immunoassay (R&D Systems) following the instructions provided in the manual.

Techniques: Immunostaining, Isolation, Expressing, Negative Control

Journal: The Journal of reproduction and development

Article Title: Expression pattern of progranulin in the human placenta and its effect on cell proliferation in the choriocarcinoma cell line BeWo.

doi: 10.1262/jrd.10-073k

Figure Lengend Snippet: Fig. 5. A–C: Studies were performed on isolated first trimester trophoblast cells. A: Secretion of progranulin in culture medium (n=4). Progranulin levels significantly increased over time (ANOVA, P<0.05). Mean ± SD. Asterisks denote significance compared with the start point at 0 h (post hoc analysis, P<0.05). B and C: No influence of migration or invasion by addition of progranulin (n=3). D: Control of proliferation revealed significant stimulation of DNA synthesis in BeWo cells, but not in Jeg-3 cells (n=3). All values are shown as means + SD. Asterisks denote significance compared with the control (post hoc analysis, P<0.05).

Article Snippet: For quantification of progranulin levels, we used the monoclonal Quantikine® human progranulin immunoassay (R&D Systems) following the instructions provided in the manual.

Techniques: Isolation, Migration, Control, DNA Synthesis

Journal: Immunity

Article Title: Signaling through the inhibitory Fc receptor FcγRIIB induces CD8 T cell apoptosis to limit T cell immunity

doi: 10.1016/j.immuni.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dr. Mark Cragg InVivoMAb anti-mouse CD16/32 (clone 2.4G2) BioXcell Cat# BE0307, RRID:AB_2736987 Bacterial and Virus Strains Biological Samples Emory Transplant Center Biorepository Emory IRB protocol #00046593 Chemicals, Peptides, and Recombinant Proteins GolgiPlug BD Biosciences Cat# 555029 OVA peptide 257–264 (SIINFEKL) GenScript Cat# RP10611 CTLA-4Ig (abatacept) Bristol-Myers Squibb, Abatacept (Orencia) Anti-CD28 domain antibodies Bristol-Myers Squibb Collagenase (type 1A) Sigma-Aldrich C2654 Hyaluronidase (type 1) Sigma-Aldrich H3506 Collagenase P Sigma-Aldrich Ref# 11213865001 Collagenase D Sigma-Aldrich Ref# 1088866001 Recombinant Fgl2 R&D Systems R&D Cat# 5257-FL-050 Critical Commercial Assays MACS CD8a+ T cell isolation kit, mouse Miltenyi Biotec 130-104-075 Quick-RNA MicroPrep Kit Zyma Research R1051 SMART-seq v4 cDNA synthesis kit Takara Cat# 634894 NexteraXT kit Illumina FC-131–1096 Mouse Fgl2 ELISA Biolegend Biolegend Cat# 437807 RNeasy Plus Micro Kit Qiagen Qiagen Cat# 74034 High Capacity cDNA reverse transcription kit ThermoFisher ThermoFisher Cat# 4368814 HT HG-U133 Plus PM BeadChip Affymetrix Thermo Fisher Cat# 901261 Lightning Link R-PE Antibody Labeling kit Novus Biologicals Cat # 703–0010 CountBright Beads Life Technologies Cat # C36950 Deposited Data RNAseq data NCBI Gene Expression Omnibus (GEO) GSE118439 Experimental Models: Cell Lines B16 Melanoma-OVA Brown, et al., 2001 , provided by Dr. Yang-Xin Fu Experimental Models: Organisms/Strains NCI C57Bl/6Ncr (NCI grantee program) Charles River Stock #556 NCI B6-LY5.1/Cr (NCI grantee program) Charles River Stock #564 OT-I Hogquist et al., 1994 OT-II Barnden et al., 1998 mOVA (C57Bl/6 background, H-2b) Ehst et al., 2013 Dr. Marc Jenkins B6.129P2-Aicda tm1(cre)Mnz /J ( Aicda −/− ) The Jackson Laboratory Stock #007770 B6;129S-Fcgr2btm1Ttk/J ( Fcgr2b −/− ) The Jackson Laboratory Stock #002848 EM:06078 Fcgr2b Fcgr2bB6null B6(Cg)-Fcgr2btm12Sjv/Cnbc ( Fcgr2b −/− ) European Mutant Mouse Archive; Dr. J.S.

Techniques: Control, Virus, Recombinant, Cell Isolation, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Antibody Labeling, Gene Expression, Mutagenesis, Software