Journal: Arthritis Research & Therapy

Article Title: Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

doi: 10.1186/s13075-021-02663-z

Figure Lengend Snippet: High-fat diet increases monocytic-MDSC expansion during PTOA. A Gating strategy used to define the M-MDSC subpopulation (CD11b + Ly6C + Ly6G − cells) in mouse bone marrow. Following the initial FS/SC discrimination, the gate was set on CD11b + cells. After exclusion of doublets (not shown), live CD11b + cells were gated and Ly6C + and Ly6G + populations. B Cytospin images of M-MDSC cells from the bone marrow from LFD and HFD mice (at 16 weeks). Scale bar denotes 10 microns. C HFD increases the BM M-MDSC population in mice ( n = 4 in each group) by total number and percentage with concomitant suppression of CD8 + and CD4 + T-cells compared to LFD mice ( n = 6 in each group, at 16 weeks). D HFD increases the BM M-MDSC population in DMM mice ( n = 3 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA Tukey’s multiple comparison test

Article Snippet: The following antibodies were added in staining buffer (eBioscience) for 30 min: CD11b-APC (clone M1/70, # 130-113-231, Miltenyi), Ly6G-PE (clone 1A8,130-102-392), and Ly6C-FITC (clone REA796, #130-111-915, Miltenyi).

Techniques: Comparison

Journal: EJHaem

Article Title: siRNA against CD40 delivered via a fungal recognition receptor ameliorates murine acute graft‐versus‐host disease

doi: 10.1002/jha2.439

Figure Lengend Snippet: Reduced graft‐versus‐host disease (GvHD) lethality in mice treated with small interfering RNA (siRNA) against CD40. (A) Experimental protocol of allogenic bone marrow transplantation (BMT): all mice received 8 Gy total body irradiation. In the allogeneic BMT group, F1 mice were given B6 donor splenocytes and BM cells. F1 mice received F1 splenocytes and BM cells to establish the syngeneic BMT group. Allogenic BMT mice were treated either with/without siRNA targeting mouse CD40 (siCD40/schizophyllan [SPG]) called NJA‐312, NJA‐302, or NJA‐515. (B) Splenocytes retrieved at day 34 after BMT were co‐stained with antibodies against Dectin1, CD40, and Ly6c. Percentages of Dectin+ and Dectin+CD11c‐ cells were determined by three‐color flourescence‐activated cell sorting (FACS) in splenocytes from mice receiving 8 Gy (irradiation, n = 2) without BMT (noBMT, 8 Gy, n = 3). (C) Splenocytes retrieved at indicated time points and treatment groups (after NJA‐312, NJA‐302, and NJA‐515 treatment) were analyzed by FACS using the following antibodies: anti‐Dectin1, anti‐Ly6c, and anti‐CD40 ( n = 1–2/group). (D) Survival of mice undergoing indicated treatments was monitored daily. Data were plotted as a Kaplan–Meier curve. Statistics using SPSS software using log‐rank (Mantel‐Cox). Left panel: indicated groups are shown (same data as left panel). The number of mice per group were as follows: *NJA‐312low ( n = 11); >80 d irradiation ( n = 12); >80 syngeneic BMT ( n = 10); d73 NJA‐302 ( n = 14); d73 NJA‐515 ( n = 15); d73 NJA‐312high ( n = 13); d34 allog BMT ( n = 25). (E) Bodyweight was determined weekly ( n = 7 per treatment group). Left panel: all groups are shown. Right panel: only indicated groups are shown (same data as left panel). * p < 0.05; ** p < 0.01, *** p < 0.0001

Article Snippet: Cells (1 × 10 6 ) were stained with Fluorescein isothiocyanate Fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse Ly6c (AbD Serotec, Duesseldorf, Germany), FITC‐conjugated H2kd, Phycoerythrin (PE)‐conjugated anti‐H2kb, PE‐conjugated anti‐CD40, PE‐conjugated anti‐CD4, FITC‐conjugated anti‐CD8a, APC‐conjugated anti‐B‐220, APC‐conjugated anti‐CD369 (PharMingen, San Diego, CA, USA), and APC‐conjugated CD3e and analyzed on a FACSCalibur.

Techniques: Small Interfering RNA, Transplantation Assay, Irradiation, Staining, FACS, Software

Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 1 Cancer-conditioned myeloid cells chronicle the evolution of pancreatic ductal adenocarcinoma (PDA) in KPC mice. (A) The number of pancreatic Treg (CD45+CD4+FoxP3+), macrophages (CD45+CD11b+F4/80+), myeloid-derived suppressor cells (MDSC) (CD45+CD11b+RB6-8C5 [Ly6G/ Ly6C]+) and NK cells (CD45+NK1.1+) for each pancreas were quantified from normal pancreas (nl), 6–8-week-old KPC pancreata with confirmed preinvasive disease (Pre) and invasive tumours (PDA). Significant differences were detected in the number of Treg, tumour-associated macrophages and MDSC during disease progression. (B) Evolving profiles of three distinct populations of myeloid cells (gated on CD45+CD11b+) were seen in various organs based on expression patterns of Gr-1 and Ly6C. BM, bone marrow; LN, lymph node. (C) The percentages (top panel) and absolute numbers (bottom panel) of CD45+CD11b+ myeloid populations in the spleen and pancreas in normal (black filled circles) and preinvasive (grey filled circles) and invasive (open circles) disease settings. Data are plotted as mean±SEM and each data point represents an individual mouse. Granulocytic MDSC (Gr-MDSC)=CD45+CD11b+Gr-1highLy6Cint; monocytic MDSC (Mo-MDSC)=CD45+CD11b+Gr-1intLy6Chigh and macrophage (Mac) =CD45+CD11b+Gr1intLy6Cint. (D) Specific immunofluorescence reveals rare Ly6G/Ly6C+ (RB6-8C5) cells in normal pancreas, focal accumulation in pancreata with preinvasive disease and diffuse infiltration in invasive PDA. Specific Ly6G immunofluorescence demonstrates that Gr-MDSC are absent from normal pancreas, rare in preinvasive disease and abundant in invasive PDA. The majority of myeloid cells in normal pancreas and surrounding preinvasive lesions appear to be macrophages. Arrowheads, epithelial cells; arrows, myeloid cells; asterisk, Gr-MDSC. Scale bars, 50 mm. (E) Ly6G/Ly6C (RB6-8C5) staining in normal pancreas and KPC salivary gland. Arrowheads, epithelial cells; arrows, myeloid cells. Scale bars, 10 mm. *p<0.05; **p<0.005; ***p<0.0005.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, Biomarker Discovery, Expressing, Staining

Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 4 Systemic administration of 1A8 (αLy6G) specifically depletes Gr-myeloid-derived suppressor cells (Gr-MDSC) in autochthonous pancreatic ductal adenocarcinoma (PDA). (A) Representative myeloid cell profiles in peripheral blood mononuclear cells from a normal mouse and untreated (KPC) and 1A8-treated KPC (KPC + 1A8) mice. Numbers indicate the percentage of each subset gated on CD45 mononuclear cells. We note that the gates for the discrete subpopulations defined in the blood were then applied to the tissue-specific analyses. (B) Percentage of Gr-MDSC (squares) and monocytic MDSC (Mo-MDSC; circles) in blood after 1A8 treatment. Data represent mean±SD from three independently treated animals. (C) Representative fluorescence activated cell sorting (FACS) profiles of CD45+ CD11b+ splenocytes from control (−) and 1A8-treated (+) KPC mice (4–6 animals per group). (D) Both the percentage and number of splenic Gr-MDSC at endpoint of 1A8 treatment (day 12) are significantly decreased (**, p=0.005). (E) Representative FACS profiles of intratumoral myeloid cells in control (−) and 1A8-treated (+) KPC mice at day 12 of Gr-MDSC depletion. (F) The percentage and number of Gr-MDSC in PDA at the endpoint are significantly decreased in 1A8-treated mice compared with control KPC mice.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, FACS, Control

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 2. Expression of mouse CST gene in various tissues. (A) Northern blot of CST mRNAs. First, 20 mg total RNA from indicated organs was electrophoresed, blotted, and hybridized with a digoxigenin-labeled (±) strand RNA probe made from the full-length mouse CST cDNA. Lane 1, kidney; lane 2, brain; lane 3, testis; lane 4, heart; lane 5, skeletal muscle; lane 6, small intestine; lane 7, stomach; lane 8, liver; lane 9, lung; lane 10, spleen. (B) RT-PCR analysis of CST and actin mRNAs. Next, 1 mg total RNA from indicated organs was reverse-transcribed with a random primer. The resulting cDNAs were separately amplified by PCR using a set of CST primers, 50-GGGTTTCCTGAGATGAC-30 (nucleotides 1±17 in Fig. 1) and 50-TAGTGCGCGTTGTAGCT-30 (nucleotides 634±650) and actin primers, 50-TTACCAACTGGGACGACATG-30 (nucleotides 149±168 in [16]) and 50-AGGAGCCAGAGCAGTAATCT-30 (nucleotides 869±888). The PCR products were separately electrophoresed, blotted, and hybridized with CST and actin probes. The observed PCR products showed the predicted sizes of 650 (CST) and 740 bases (actin). Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4, kidney; 5, stomach; 6, small intestine; 7, lung; 8, liver; 9, spleen; 10, testis. (C) CST activity. Whole homogenates from indicated organs were assayed for CST activity and protein concentration as described in Materials and methods. 1, kidney; 2, brain; 3, testis; 4, heart; 5, skeletal muscle; 6, small intestine; 7, stomach; 8, liver; 9, lung; 10, spleen.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Activity Assay, Protein Concentration

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 3. Multiple forms of mouse CST cDNA with 50-UTR unique sequences. Seven types of unique DNA sequences on the 50 end of mouse CST cDNAs are represented by boldface letters. The 50-terminus of the homologous sequence is indicated by arrows. The translation-initiation codon ATG is boxed and indicated by arrowheads. In forms A, B2, and C, type a, b, and c sequences are underlined with continuous lines and type d sequence is underlined with broken lines.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Sequencing

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 5. RT-PCR analysis of tissue-specific use of alternative exon 1. (A) Position of each primer and probe. Boxes represent exons. Symbols for exons are the same as in Fig. 4. Black boxes are coding exons. Total RNAs from various tissues were reverse transcribed using GSP1 primer. After isolation of the first cDNA strand, PCR was performed using a combination of a common antisense-primer 2A, corresponding to exon 2, and seven sense-primers relating to the seven exons 1, aS, bS, cS, dS, eS, fS, or gS. The PCR products were subjected to Southern hybridization with a probe EX2, corresponding to exon 2. (B) RNA from indicated organs was examined. PCR was performed using sense-primer aS (lane a), bS (lane b), cS (lane c), dS (lane d), eS (lane e), fS (lane f ), or gS (lane g). Minor bands in lanes a and b are represented by asterisks in small intestine.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, Hybridization

Journal: Cell

Article Title: Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG3

doi: 10.1016/j.cell.2018.11.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Mouse Ly6C (clone HK1.4), 162-Dy , Fluidigm , Cat#: 3162014B.

Techniques: Functional Assay, Virus, Recombinant, Mass Cytometry, Conjugation Assay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, cDNA Library Assay, Software