arg1  (Proteintech)

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Analysis of macrophage activation in the uterine tissue of cows with endometritis. (A, B) Representative immunofluorescence (IF) staining images (A) and quantitative analysis (B) of iNOS (M1 marker, red) in endometrial tissues from healthy cows and cows with endometritis. Nuclei were counterstained with DAPI (blue). (C, D) Representative IF staining images (C) and quantitative analysis (D) of Arg1 (M2 marker, red) in endometrial tissues. (E, F) Relative mRNA expression levels of iNOS (E) and Arg1 (F) in endometrial tissues, as determined by qPCR. (G, H) Relative expression levels of IL-1β, IL-6 and TNF-α in endometrial tissues, as determined by IHC. (I, J) Relative mRNA expression levels of IL-1β (I) and IL-6 (J) in endometrial tissues, as determined by qPCR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Activation Assay, Immunofluorescence, Staining, Marker, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Exosomes from LPS-stimulated EECs induce pro-inflammatory macrophage activation. (A) Schematic diagram of the experimental setup for exosome uptake. (B) Fluorescence microscopy images showing the uptake of PKH67-labeled exosomes (green) by macrophages. Cytoskeleton was stained with Phalloidin (red), and nuclei were stained with DAPI (blue). (C) Western blotting analysis of phosphorylated NF-κB p65 (p-p65) in macrophages treated with Control-exo or LPS-exo. (D, E) Representative immunofluorescence (IF) staining images (D) and quantitative analysis (E) of iNOS (greed) in macrophages. (F, G) Representative IF staining images (F) and quantitative analysis (G) of Arg1 (red) in macrophages. (H) Schematic diagram of co-culture experiments. (I, J) Relative mRNA expression levels of iNOS (I) and Arg1 (J) in macrophages after co-culture with EECs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Activation Assay, Fluorescence, Microscopy, Labeling, Staining, Western Blot, Control, Immunofluorescence, Co-Culture Assay, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Transcriptomic profiling reveals significant enrichment of lncRNA OTUD6B-AS1 in exosomes derived from LPS-stimulated EECs. (A, B) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative expression levels of iNOS (A) and Arg1 (B) in macrophages. (C) Schematic overview of the RNA sequencing and analysis workflow. (D) Volcano plot showing differentially expressed lncRNAs in LPS-exo compared to Control-exo. (E, F) Gene Ontology (GO) biological process enrichment analysis (E) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (F) of the differentially expressed lncRNAs. (G) qPCR validation of the 6 upregulated lncRNAs in Control-exo and LPS-exo. (H) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages. (I) A proposed competing endogenous RNA (ceRNA) network involving lncRNA OTUD6B-AS1, miR-128, and Notch2. (J) Relative mRNA expression level of lncRNA OTUD6B-AS1 in control and LPS-stimulated EECs. (K, L) RNA fluorescence in situ hybridization (RNA-FISH) showing the subcellular localization of lncRNA OTUD6B-AS1 (red) in EECs (K) and its quantitative cytoplasmic/nuclear distribution (L). Nuclei were stained with DAPI (blue). (M – P) Relative mRNA expression levels of lncRNA OTUD6B-AS1 (M − O) and miR-128 (P) in endometrial tissues from healthy cows and cows with endometritis, as determined by qPCR (O, P) and RNA-FISH (M) with quantification (N). (Q) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 protein levels in endometrial tissues. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Derivative Assay, Incubation, Expressing, RNA Sequencing, Control, Biomarker Discovery, Fluorescence, In Situ Hybridization, Staining, Western Blot

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: EECs-derived exosomes induce pro-inflammatory macrophage activation via delivery of lncRNA OTUD6B-AS1. (A) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages treated with Control-exo or LPS-exo. (B, C) RNA-FISH images (B) and quantitative analysis (C) showing lncRNA OTUD6B-AS1 (red) transfer to macrophages after co-culture with Control-exo or LPS-exo. Nuclei were stained with DAPI (blue). (D) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages after transfection with lncRNA OTUD6B-AS1 overexpression plasmids (OE-lncRNA) or control plasmids (OE-NC). ( E – I) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (E), along with immunofluorescence (IF) quantitative analysis of iNOS (F, G) and Arg1 (H, I) protein levels in macrophages after transfection with OE-lncRNA or OE-NC. (J – N) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (J), along with IF quantitative analysis of iNOS (K, L) and Arg1 (M, N) protein levels in macrophages after lncRNA OTUD6B-AS1 knockdown (si-lncRNA) or control treatment (si-NC). (O) Relative mRNA expression level of lncRNA OTUD6B-AS1 in exosomes isolated from lncRNA OTUD6B-AS1-knockdown LPS-stimulated EECs (si-lncRNA-LPS-exo) or exosomes from siRNA NC-transfected LPS-stimulated EECs (si-NC-LPS-exo). (P – S) IF quantitative analysis of iNOS (P, Q) and Arg1 (R, S) protein levels in macrophages treated with si-lncRNA-LPS-exo or si-NC-LPS-exo. (T) Relative mRNA expression levels of iNOS and Arg1 in macrophages treated with exosomes isolated from control EECs overexpressing lncRNA OTUD6B-AS1 (OE-lncRNA-Control-exo) or exosomes from control plasmids-transfected EECs (OE-NC-Control-exo). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Derivative Assay, Activation Assay, Expressing, Control, Co-Culture Assay, Staining, Transfection, Over Expression, Western Blot, Immunofluorescence, Knockdown, Isolation

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: lncRNA OTUD6B-AS1 acts as a ceRNA by sponging miR-128 to facilitate pro-inflammatory macrophage activation. (A) Relative mRNA expression level of miR-128 in macrophages treated with Control-exo or LPS-exo. (B) Luciferase reporter assay in HEK293T cells co-transfected with wild-type (WT) or mutant (MUT) lncRNA OTUD6B-AS1 reporter plasmids and miR-128 mimic or mimic NC. (C) RNA pull-down detection of the enrichment of miR-128 to lncRNA OTUD6B-AS1. (D) Ago2 RIP assay analysis of the enrichment of lncRNA OTUD6B-AS1 pulled-down from the Ago2 protein. (E) Relative mRNA expression level of miR-128 in macrophages transfected with OE-NC or OE-lncRNA. (F – J) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (F), along with immunofluorescence (IF) quantitative analysis of iNOS (G, H) and Arg1 (I, J) protein levels in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. (K, L) Relative mRNA expression levels of IL-1β (K) and IL-6 (L) in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Activation Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Mutagenesis, Western Blot, Immunofluorescence

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Notch2 mediates the regulatory effect of the lncRNA OTUD6B-AS1/miR-128 axis on macrophage activation. (A) Predictive analysis of miR-128 targets using multiple databases. (B) Western blotting analysis of Notch2 protein levels in macrophages treated with Control-exo or LPS-exo. (C) Western blotting analysis of Notch2 protein levels in macrophages transfected with OE-NC or OE-lncRNA. (D – H) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (D), along with immunofluorescence (IF) quantitative analysis of iNOS (E, F) and Arg1 (G, H) protein levels in macrophages treated with OE-NC or OE-lncRNA and the Notch2 inhibitor DAPT. (I) Luciferase reporter assay in HEK293T cells co-transfected with WT or MUT Notch2 3′UTR reporter plasmids and miR-128 mimic or mimic NC. (J, K) Relative protein (J) and mRNA (K) expression levels of Notch2 in macrophages transfected with miR-128 mimic or mimic NC. (L – P) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (L), along with IF quantitative analysis of iNOS (M, N) and Arg1 (O, P) protein levels in macrophages co-treated with miR-128 inhibitor or inhibitor NC and DAPT. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The antibodies used include iNOS (Proteintech, Cat # 18985-1-AP), Arg1 (Proteintech, Cat # 18985-1-AP), Goat Anti-Rabbit IgG (Bioss, Cat # bs-0295G) and Goat Anti-Mouse IgG (Bioss, Cat # bs-0296Gs).

Techniques: Activation Assay, Western Blot, Control, Transfection, Immunofluorescence, Luciferase, Reporter Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: GFPT2 promotes macrophage dysfunction in atherosclerosis via ACADL glycosylation-dependent suppression of ACADL and Arg1

doi: 10.1007/s00018-026-06203-3

Figure Lengend Snippet: GFPT2 mediated ACADL glycosylation to regulate Arg1 expression. ( A – B ) qRT-PCR and WB analysis of ACADL expression in abdominal aortic tissue and primary peritoneal macrophages from AS mice. ( C ) IF double staining showing co-localization of GFPT2 and ACADL in mouse BMDMs with or without ox-LDL treatment. ( D ) WB analysis of ACADL expression in ox-LDL-treated RAW264.7 cells. ( E ) Deglycosylation assay confirming N-glycosylation of ACADL using PNGase F kit. ( F ) WB analysis was performed to examine the effect of ACADL-N273 glycosylation site mutation on ACADL expression. ( G ) WB assay was performed to evaluate the effect of GFPT2 knockdown on ACADL protein level. ( H–L ) WB and IF analyses demonstrating the influence of glycosylation inhibitor TM treatment on GFPT2 overexpression regulating ACADL and Arg1 expression in ox-LDL-treated RAW264.7 cells or mouse BMDMs. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. Con/ Ctrl/ ox-LDL + sh-NC/ ox-LDL + OE-NC/ ox-LDL + OE-GFPT2

Article Snippet: Nuclei were counterstained with DAPI for 5 min. For IF staining of cellular ACADL, Arg1, or phosphorylated NF-κB subunit p65 (p-p65), RAW264.7 macrophages or BMDMs were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 5% normal goat serum for 30 min, cells were incubated with primary antibodies against ACADL (#17526-1-AP, 1:200; Proteintech), Arg1 (#DF6657, 1:200; Affinity Biosciences, Liyang, Jiangsu, China), or p-p65 (#HY- P80839 , 1:200; MCE) overnight at 4 °C.

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Double Staining, Mutagenesis, Knockdown, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: GFPT2 promotes macrophage dysfunction in atherosclerosis via ACADL glycosylation-dependent suppression of ACADL and Arg1

doi: 10.1007/s00018-026-06203-3

Figure Lengend Snippet: GFPT2 regulated Arg1 expression to influence foam cell formation, macrophage efferocytosis, and pro-inflammatory responses. ( A – B ) qRT-PCR and WB analysis of Arg1 expression in abdominal aortic tissues and primary peritoneal macrophages from AS mice. ( C – D ) Assessment of Arg1 mRNA and protein levels in ox-LDL-treated RAW264.7 macrophages. ( E – F ) Effect of GFPT2 knockdown on Arg1 expression in ox-LDL-treated RAW264.7 macrophages. ( G – H ) Oil Red O staining demonstrating the effect of modulating the GFPT2-Arg1 axis on intracellular lipid accumulation in RAW264.7 macrophages. ( I ) Representative confocal microscopy images showing the impact of GFPT2-Arg1 axis regulation on cellular uptake of Dil-labeled ox-LDL, as quantified by fluorescence intensity in RAW264.7 macrophages. ( J – K ) Fluorescence microscopic analysis illustrating the effect of GFPT2-Arg1 axis modulation on RAW264.7 macrophage engulfment of apoptotic thymocytes (green: labeled apoptotic thymocytes; red: RAW264.7 macrophages). ( L – P ) Flow cytometry and qRT-PCR analysis of the GFPT2-Arg1 axis influence on RAW264.7 macrophage surface receptors CD36 and MerTK. ( Q – R ) ELISA quantification showing the regulatory effects of GFPT2-Arg1 axis manipulation on MCP-1 and IL-8 levels in RAW264.7 macrophage culture supernatants. ***p < 0.001, **p < 0.01, *p < 0.05 vs Con/ Ctrl/ ox-LDL+sh-NC/ ox-LDL+sh-GFPT2+DMSO

Article Snippet: Nuclei were counterstained with DAPI for 5 min. For IF staining of cellular ACADL, Arg1, or phosphorylated NF-κB subunit p65 (p-p65), RAW264.7 macrophages or BMDMs were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 5% normal goat serum for 30 min, cells were incubated with primary antibodies against ACADL (#17526-1-AP, 1:200; Proteintech), Arg1 (#DF6657, 1:200; Affinity Biosciences, Liyang, Jiangsu, China), or p-p65 (#HY- P80839 , 1:200; MCE) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Staining, Confocal Microscopy, Labeling, Fluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: GFPT2 promotes macrophage dysfunction in atherosclerosis via ACADL glycosylation-dependent suppression of ACADL and Arg1

doi: 10.1007/s00018-026-06203-3

Figure Lengend Snippet: GFPT2 knockdown ameliorated AS in vivo. ( A – B ) Body weight monitoring and serum lipid profile measurement, including TC, TG, LDL-C and HDL-C, revealing the effects of GFPT2 knockdown on weight gain and lipid profile change in AS mice. ( C – D ) WB and IF analyses were performed to assess the impact of GFPT2 knockdown on the expression of GFPT2, ACADL, and Arg1 in abdominal aortic tissues of AS mice. ( E ) IF double staining demonstrating the impact of GFPT2 knockdown on the co-localization of the macrophage marker CD68 with GFPT2 or ACADL. ( F – I ) H&E and Oil-red-O staining evaluating the effects of GFPT2 knockdown on plaque formation, foam cell accumulation, and lipid deposition in abdominal aortic tissues. ( J – K ) In vivo, fluorescence microscopy was employed to examine the influence of GFPT2 knockdown on efferocytosis function of primary peritoneal macrophages. ( L – R ) Flow cytometry, qRT-PCR and ELISA assays demonstrated that GFPT2 knockdown modulated CD36 and MerTK expression in surface of primary peritoneal macrophages from AS mice, as well as MCP-1 and IL-8 serum levels. ***p < 0.001, **p < 0.01, *p < 0.05 vs Con/ AS+sh-NC

Article Snippet: Nuclei were counterstained with DAPI for 5 min. For IF staining of cellular ACADL, Arg1, or phosphorylated NF-κB subunit p65 (p-p65), RAW264.7 macrophages or BMDMs were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 5% normal goat serum for 30 min, cells were incubated with primary antibodies against ACADL (#17526-1-AP, 1:200; Proteintech), Arg1 (#DF6657, 1:200; Affinity Biosciences, Liyang, Jiangsu, China), or p-p65 (#HY- P80839 , 1:200; MCE) overnight at 4 °C.

Techniques: Knockdown, In Vivo, Expressing, Double Staining, Marker, Staining, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A to I ) Serpine1 +/+ and Serpine1 −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected at day 21. [(A) and (B)] Flow cytometry quantification of frequencies of total T cells (CD45 + CD3 + ) (A) and conventional dendritic cells (CD45 + F4/80 − CD11c + MHCII + ) (B) among live cells in orthotopic tumors. (C) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). (D) Quantitative RT-PCR analysis of Arg1 in BMDMs treated with vehicle or recombinant PAI1 (rPAI1) for 20 hours. Each dot represents individual primary BMDM lines. (E) Flow cytometry quantification of frequency of CD8 + T cells (CD45 + CD3 + CD8 + ) among live cells in orthotopic tumors. [(F) and (G)] Flow cytometry quantification of frequencies and absolute numbers of GZMB + CD8 + T cells (F) and Ki67 + CD8 + T cells (G) following a 5-hour ex vivo PMA/ionomycin stimulation of tumor digests. (H) Co-IF staining for GZMB (green), CD8 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). (I) Co-IF staining for Ki67 (red), CD8 (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( J ) Experimental design for CD8 + T cell depletion. Mice received control immunoglobulin G (IgG) or anti-CD8 antibody twice per week, starting 5 days postimplantation (7940B). Tumors were harvested at day 21. ( K ) Tumor weight of orthotopic tumors with CD8⁺ T cell antibody-mediated depletion. Symbols in (A) to (I) and (K) represent individual mice. Data are means ± SEM. P values were determined by two-tailed unpaired t test [(A), (B), right of (C), (D), (E) to (G), and right of (H)], Mann-Whitney test [left of (C), left of (H), and (I)], and Mann-Whitney test with Holm-Sidak post hoc (K).

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Flow Cytometry, Staining, Quantitative RT-PCR, Recombinant, Ex Vivo, Control, Two Tailed Test, MANN-WHITNEY

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. Plat +/+ and Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from Plat +/+ and Plat −/− mice. ( C ) ELISA of tPA in tissue supernatants from pancreata of Plat +/+ mice (no tumor) and orthotopic tumors from Plat +/+ and Plat −/− mice. ( D ) Picrosirius red staining of orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( E ) Co-IF staining for cCasp3 (red), ECAD (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 4 FOV per animal). ( F to H ) Flow cytometry quantification of frequencies of total T cells (F), CD8 + T cells (G), and conventional dendritic cells (H) among live cells in orthotopic tumors. ( I ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (I) represent individual mice. Data are means ± SEM. P values were determined by Mann-Whitney test [(B) and (F)], two-way ANOVA with Holm-Sidak post hoc (C), and two-tailed unpaired t test [(D), (E), and (G) to (I)].

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice. ( C ) IHC staining for CD8 in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). ( D ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (D) represent individual mice. Data are means ± SEM. P values were determined by one-way ANOVA with Holm-Sidak post hoc [(B) to (D)]. ( E ) Working model. Left: Hypoxia stabilizes HIF proteins, inducing PAI1 expression in pancreatic CAFs. Although tPA levels are elevated in PDAC, PAI1 predominantly inhibits its activity, thereby suppressing antitumor immunity. Middle: Elimination of stromal PAI1 restores tPA activity, enhancing antitumor CD8 + T cell responses, accompanied by alleviation of immunosuppressive TAM phenotypes and increased dendritic cell (DC) infiltration. Stromal PAI1-driven tumor growth depends on CD8 + T cells. Right: Removal of stromal tPA abolishes residual tPA activity, further promoting an immunosuppressive TME and tumor growth.

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay