Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 1. The expressions of PD-L1 in human preeclampsia (PE) placentas (n = 30) and normal pregnant women (n = 30). (A,B) PD-1 levels were significantly reduced in PE placentas. (C,D) PD-L1 levels were overtly reduced in pre-eclamptic mothers. (E,F) GM-CSF levels were prominently decreased in the PE set. Relative JAK2 (G) and STAT5 (H) protein levels were markedly lessened, while those of p-JAK2 (I) and p-STAT5 (J) were significantly enriched in pre-eclamptic placentas. (K) Western blotting results of related molecules were shown. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 2. The transfection efficiency in cell lines and the changes in expressions of relevant factors after lentivirus treatment (n = 3, for each group). (A,B) The valid transfection efficiency in the two cell lines was confirmed by the presence of green fluorescence. Scale bars, 100 μm. The mRNA (C,L) and protein (D,M) levels of PD-L1 were significantly reduced after the knockdown of PD-L1 in two cell systems. The mRNA (E,N) and protein (F,O) levels of GM-CSF were notably lower after transfection with siR-PD-L1 in the two trophoblasts. JAK2 and STAT5 protein expressions were induced, while their phosphorylation expressions were inhibited in HTR- 8/SVneo (G–J) and JEG-3 (P–S) cells with the introduction of LV-PD-L1. (K,T) Western blot of PD-L1, GM- CSF, and JAK2/STAT5 molecules was carried out in the two cell lines. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Transfection, Fluorescence, Knockdown, Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 4. Influences of aberrant GM-CSF on PD-L1 and the JAK2/STAT5 pathway (n = 3, for each group). The protein levels (A,H) of PD-L1 were evidently elevated after adding GM-CSF in trophoblasts. GM-CSF protein levels (B,I) were significantly higher by the accumulation of GM-CSF in the two trophoblasts. The levels of JAK2 and STAT5 were overtly lower, while their phosphorylation levels were enhanced exposure to the GM- CSF neutralizing antibody in HTR-8/SVneo (C–F) and JEG-3 (J–M) cells. (G,N) Western blot bands were displayed in the two cell lines. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 5. The biological role of GM-CSF in the cell lines (n = 3, for each group). (A,B)Over-regulation of GM- CSF elevated the migratory property of cells. (C,D) GM-CSF stimulation increased cell invasion. Scale bar, 500 μm. (E,F) GM-CSF up-regulation suppressed cell apoptosis. (G–I) Increased GM-CSF promoted the evolvement of cell cycle from the G1 to the S stage in both cells. (J) (K) Cells supplemented with GM-CSF exhibited higher proliferative ability in trophoblasts. ns, no significance. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques:

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 6. The rescue assay and the impact of the JAK2 inhibitor on regulating GM-CSF (n = 3, for each group). (A,B) The rescue assay of co-transfection of PD-L1 and GM-CSF in two kinds of trophoblasts. The protein levels (C,H) of GM-CSF were elevated with the JAK2 inhibitor. JAK2 (D,I) and STAT5 (E,J) protein levels were not influenced by the JAK2 inhibitor. The levels of p-JAK2 (F,K) and p-STAT5 (G,L) were profoundly inhibited by the addition of JAK2 inhibitor. (M) The bands of western blot in rescue experiments. (N) Western blotting was shown in cells with the JAK2 inhibitor. ns, no significance. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Rescue Assay, Cotransfection, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 7. The blood pressure, urine protein, and the expressions of related indicators in different groups of pregnant rats (n = 5, for each group). (A,B) LV.PD-L1 reduced the blood pressure of PE-like animals on GD17 and GD19. (C) LV.PD-L1 significantly decreased the urinary protein content on GD19. Fetal weight (D), fetal length (E), and placental weight (F) in different groups. (G) Western blot in rat placental tissues. (H,I) The expressions of PD-1 were evidently reduced after L-NAME treatment. (J,K) The levels of PD-L1 were notably decreased in placentas from PE-like pregnant rats. (L,M) The expressions of GM-CSF were elevated following adding LV.PD-L1. (N–Q) The relative band intensity of the JAK2/STAT5 pathway was compared using quantification. DAPI staining and the green fluorescence representing viral infection were displayed in the L-NAME + PD-L1 NC (R) and L-NAME + LV.PD-L1 (S) sets. Scale bar, 50 μm. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Western Blot, Staining, Fluorescence, Infection

Journal:

Article Title: Identification of CD4 + T Cell Epitopes from NY-ESO-1 Presented by HLA-DR Molecules

doi:

Figure Lengend Snippet: A, Purification of full-length NY-ESO-1 protein using a Ni2+ chromatography column. SDS polyacrylamide gel showed the crude extract from E. coli strain BL21(DE3) bearing pET28 vector (lane 1), pNY-ESO-1 (lane 2), the purified NY-ESO-1 protein (lane 3), bacterial extract encoding the truncated NY-ESO-1 (lane 4), and the purified truncated NY-ESO-1 protein, ESO1–74 (lane 5). B, Western blot to confirm the specificity of Abs against NY-ESO-1. Sera at 1/2000 dilution from two representative patients, one with (lanes 1–3) and one without (lanes 4–6) detectable NY-ESO-1 Abs by ELISA, were used against bacterial extract encoding the vector only (lanes 1 and 4), encoding NY-ESO-1 (lanes 2 and 5), and the purified NY-ESO-1 protein (lanes 3 and 6). C, Patient TE was among the melanoma patients who had Abs against NY-ESO-1 protein. ELISA was performed using sera from 88 patients for the presence of Abs against NY-ESO-1 (BSA as control protein). Values of OD 450 at 1/25, 1/250, and 1/2500 of sera dilutions were plotted. Sera from normal donors were used as controls, and their mean OD value was also plotted (ND).

Article Snippet: After 4-h incubation, cells were washed in serum-free RPMI medium, and ∼3 × 10 4 target cells were incubated with the same number of TE4-1 cells overnight, and cytokine release was measured using GM-CSF ELISA kits (R&D Systems, Minneapolis, MN) for human or IFN- γ kits (Endogen, Woburn, MA) for mouse.

Techniques: Purification, Chromatography, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Mediators of Inflammation

Article Title: IL-1 β and TNF α Promote Monocyte Viability through the Induction of GM-CSF Expression by Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.1155/2014/241840

Figure Lengend Snippet: Effect of IL-1 β and TNF α on M-CSF, IL-34, and GM-CSF expression in RA SF . SF were stimulated by IL-1 β (25 ng/mL) or TNF α (25 ng/mL) for 24 hours. (a)–(c) RNA was harvested and a qPCR for M-CSF, IL-34, and GM-CSF expression was performed. Results are given as the relative expression using hHPRT gene as housekeeping gene. The bars represent the mean and each plots one biological replicate ( n = 6 patients). (d) GM-CSF concentration (pg/mL) was assessed by ELISA. Results are given as the mean (± SEM). * P < 0.05; ** P < 0.001; *** P < 0.0001.

Article Snippet: GM-CSF levels were measured in RA SF conditioned media by ELISA assay (Human GM-CSF DuoSet ELISA development kit, R&D Systems).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet:

Article Snippet: Human GM-CSF ELISA Kit , R&D Systems , #DY215-05.

Techniques: Virus, Microarray, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Plasmid Preparation, Expressing, Software

Journal: Blood

Article Title: Granulocyte/macrophage–colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects

doi: 10.1182/blood-2009-05-155689

Figure Lengend Snippet: Presence of GM-CSF autoantibodies in healthy subjects. (A) Total IgG was isolated from the serum of healthy control subjects (HC) or patients with PAP (PAP) or from pharmaceutical grade IVIG by protein G chromatography and subjected to ultrafiltration under acidic conditions to remove bound GM-CSF. GM-CSF autoantibodies were then isolated by GM-CSF affinity chromatography and evaluated by far-Western analysis probed with 125I-GM-CSF. Shown are Coomassie Blue–stained electrophoresis gels (bottom panels) and corresponding far-Western blots (top panels). Each numbered lane represents the corresponding bound (left panels) and unbound (right panels) chromatography fractions from 1 subject or sample. (B) Serum GM-CSF–binding proteins from 6 healthy human subjects were fractionated individually on gels as in panel A, and proteins in the 180-kDa band from each were extracted, subjected to liquid chromatography and tandem mass spectroscopy, and the results evaluated by comparison to Mascot database as described under “Methods.” Only matches with a probability-based Mowse score greater than 64 (indicating a P value < .05) were considered in the analysis. The percentages of immunoglobulin (Ig) and non-Ig peptides among the top 50 peptide fragment matches identified for each sample are shown. (C) GM-CSF autoantibodies were isolated by GM-CSF affinity chromatography from serum of healthy subjects (HC; n = 10), patients with PAP (PAP, n = 4), or from pharmaceutical IVIG (n = 1 clinical grade vial), and the percentage of IgG subtypes was measured by ELISA. (D) Serum GM-CSF autoantibody concentrations in healthy subjects (HC), patients with PAP (PAP), or IVIG (reconstituted at 9.94 mg/mL in PBS). Serum GM-CSF autoantibody levels in healthy subjects (median (interquartile range [IQR]) = 1.04 [0.63-1.7] μg/mL, n = 72) were lower than in patients with PAP (median [IQR] = 59.8 (27.4–116.5) μg/mL; n = 21; P < .001). Median values (HC, PAP) are indicated by a horizontal bar. (E) Serum GM-CSF autoantibody levels in males (n = 15) and females (n = 57). Data are shown as whisker plots indicating the interquartile range (upper and lower borders of box), the 90th and 10th percentile (error bars), the 95th and 5th percentile (upper and lower open symbols), the median (solid horizontal line in box), and mean (dashed line in box) values of GM-CSF autoantibody levels. (F) Serum GM-CSF autoantibody levels in healthy women (△) and men ( □ ) of various ages (n = 72). Regression analysis did not reveal a significant correlation GM-CSF autoantibody levels with age (R2 = 0.08).

Article Snippet: Serum concentrations of free GM-CSF (ie, not bound to GM-CSF autoantibody) were measured using Quantikine HS Human GM-CSF ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Isolation, Control, Chromatography, Affinity Chromatography, Western Blot, Staining, Electrophoresis, Binding Assay, Liquid Chromatography, Tandem Mass Spectroscopy, Comparison, Enzyme-linked Immunosorbent Assay, Whisker Assay

Journal: Blood

Article Title: Granulocyte/macrophage–colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects

doi: 10.1182/blood-2009-05-155689

Figure Lengend Snippet: Concentration of free and IgG-bound GM-CSF in human serum. (A) Total IgG was isolated individually from the sera of healthy subjects (HC) or patients with PAP (PAP) using protein G and evaluated by Western blotting to detect GM-CSF (top panels) or IgGκ (κ) and -λ (λ) light chains (as a loading control, bottom blots). Each lane represents one subject. (B) Detection of free GM-CSF and autoantibody-bound GM-CSF in serum. A set of “standard” samples composed of recombinant human GM-CSF (Leukine) at various concentrations ranging from 0 to 30 ng/mL were prepared in mouse serum in the absence (○) or presence (▽) of purified human GM-CSF autoantibodies (30 μg/mL). Standard samples were diluted 1/30 with 1% BSA in PBS and then GM-CSF was measured using a commercial human ELISA kit (R&D Systems) as directed by the manufacturer. (C) Use of a novel ELISA (SDS-HD ELISA, see “Methods”) to quantify GM-CSF in PBS in the absence or presence of GM-CSF autoantibody (1 μg/mL) and in the absence or presence of a pretreatment with SDS and heat denaturation. Each bar represents the mean of duplicate determinations for 1 of 4 separate experiments with similar results. (D) GM-CSF level evaluated using a novel human GM-CSF ELISA as described in “Methods.” Symbols represent the same samples and conditions as described in the legend to panel B above. GM-CSF was detectable in the absence of GM-CSF autoantibody (○), undetectable in the presence of GM-CSF autoantibody in the absence of SDS-HD pretreatment (▽), and detection was restored in the presence of GM-CSF autoantibody by SDS-HD pretreatment (△). (E) Free GM-CSF (▨) or total GM-CSF (free and autoantibody-bound; ■) were measured in sera of healthy subjects (HC) or patients with PAP (PAP) using a commercially available ELISA or the SDS-HD ELISA, respectively, as described in “Methods.” Total serum GM-CSF levels in HC and PAP were not different (3048 ± 484, n = 11; PAP 2360 ± 668, n = 5; respectively, P = .43).

Article Snippet: Serum concentrations of free GM-CSF (ie, not bound to GM-CSF autoantibody) were measured using Quantikine HS Human GM-CSF ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Concentration Assay, Isolation, Western Blot, Control, Recombinant, Purification, Enzyme-linked Immunosorbent Assay