Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Changes in CCL5 and HMGB1 protein levels changes at lesion sites following rat SCI. (A) ELISA measurement of CCL5 protein levels at lesion sites following SCI at 0 d, 1 d, 4 d and 7 d, respectively. (B) Western blot analysis of HMGB1 expression following SCI at 0 d, 1 d, 4 d and 7 d. (C) Quantification data are shown in ( B ). Quantities were normalized to endogenous β-actin. (D) ELISA analysis of CCL5 protein levels at lesion sites at 0 d, 1 d, 4 d and 7 d following with or without intrathecal injection of 10 µl of an HMGB1 neutralizing antibody (HMGB1 Ab, 50 µg/kg). (E) Immunofluorescence staining revealed the colocalization of CCL5 with GFAP-positive astrocytes before or after SCI at 4 d with or without the intrathecal injection of 10 µl of HMGB1 Ab (50 µg/kg). The rectangle indicates the region magnified. Arrowheads indicate positive signals. n = 6. Scale bars, 50 μm in (E) . The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Immunofluorescence, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Examination of CCL5 production in astrocytes following stimulation with rHMGB1. ( A , B ) Purified primary astrocytes were stained with GFAP and Hoechst 33,342, and the purity was greater than 90%. Scale bar, 50 μm. ( C , D ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following stimulation with 0–2.5 µg/mL rat recombinant HMGB1 (rHMGB1) for 24 h, respectively. ( E , F ) ELISA assay was used to determine the production of CCL5 in the lysates and supernatants following primary astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of 2.5 µg/ml HMGB1 Ab, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of RAGE, TLR-2, or TLR-4 interference on HMGB1-induced CCL5 production in astrocytes. ( A , B ) Cell lysates and supernatants were tested by ELISA for the production of CCL5, following astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of the RAGE inhibitor FPS-ZM1 (2 µM) for 24 h. ( D , E ) ELISA was used to determine CCL5 protein levels in lysates and supernatants from astrocytes after stimulation with 0.5 µg/ml rHMGB1 in the presence of the TLR2 inhibitor C29 (10 µM) for 24 h. ( G , H ) CCL5 levels in lysates and supernatants of astrocytes were determined by ELISA after the astrocytes were treated with the TLR4 inhibitor TAK-242 (2 µM) in the presence of 0.5 µg/ml rHMGB1 for 24 h. ( C , F , I ) CCK8 assay of the effects of FPS-ZM1, C29, or TAK-242 on the viability of astrocytes. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Determination of CCL5 synthesis-related protein levels in astrocytes following stimulation with rHMGB1. (A) Western blot analysis of the phosphorylation of the ERK, JNK, P38 kinase and p65NF-κB proteins after astrocytes were treated with 0–2.5 µg/mL rHMGB1 for 24 h. (B-E) Quantification data are shown in (A). Quantities were normalized to endogenous β-actin. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of the inhibition of MAPK/NF-κB signaling on the HMGB1-induced production of CCL5 from astrocytes. ( A , B ) ELISA was used to determine the effects of 10 µM ERK inhibitor PD98059, 10 µM JNK inhibitor SP6001251 or 10 µM P38 inhibitor SB203580 in the presence of 0.5 µg/ml rHMGB1 for 24 h on CCL5 protein production levels in lysates and supernatants of astrocytes. (C) Western blot analysis of the activation of p65NFκB protein after astrocyte treatment with 10 µM ERK inhibitor PD98059 or 10 µM JNK inhibitor SP6001251 for 24 h in the presence of 0.5 µg/ml rHMGB1. (D) Quantification data are shown in (C). Quantities were normalized to endogenous β-actin. ( E , F ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following challenge with 10 µM SN50 for 24 h in the presence of 0.5 µg/ml rHMGB1, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-mediated astrocytic CCL5 on the migration of BV2 microglia cells or RAW 264.7 macrophage cells in vitro. (A) Illustration of the BV2 or RAW 264.7 cells with rCCL5 coculture model. (B) Transwell assay analysis of migration the ability of BV2 or RAW 264.7 cells co-incubated with 0.1 µg/ml rCCL5 for 48 h. ( C ) and ( D ) Quantification data are shown in (B). (E) Illustration of the BV2 or RAW 264.7 cells and ACM coculture model. (F) Migration assay of BV2 or RAW 264.7 cells cocultured with ACM. ACM were prepared from astrocytes exposed to 0.5 µg/ml rHMGB1 for 24 h, followed by the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. (G) and (H) Quantification data are shown in (F). Scale bars, 100 μm in ( B ) and ( F ). The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Migration, In Vitro, Transwell Assay, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-induced astrocytic CCL5 on microglia/macrophage polarization. ( A ) and ( B ) qRT-PCR was used to determine the expression of M1 markers ( CD86 , iNOS and TNF-a ) and M2 markers ( CD206 , Arg1 and Ym1 ) after BV2 or RAW 264.7 cells incubated with 0.1 µg/ml rCCL5 for 24 h. ( C ) and ( D ) The expression levels of M1 and M2 markers were determined by qRT-PCR following BV2 or RAW 264.7 cells cocultured with ACM for 24 h. ACM was prepared by astrocytes challenge with 0.5 µg/ml rHMGB1 for 24 h, after which the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Immunofluorescence of microglia/macrophage and locomotor function assessment after the inhibition of HMGB1 or CCL5 by neutralizing antibodies in rat SCI. ( A ) Immunofluorescence of IBA1- or CD68-positive cells at 4 d following SCI after neutralizing antibody application by intrathecal injection of 10 µl of neutralizing antibodies (HMGB1 Ab, 50 µg/kg), CCL5 (CCL5 Ab, 50 µg/kg) or normal rabbit IgG (IgG, 50 µg/kg). n = 6. Scale bars, 50 μm. ( B , C ) Quantitative analysis of the intensity of IBA1- or CD68-labeled cells in ( A) , respectively. (D) HE staining of the injured spinal cord at 21 d after administration of 10 µl of HMGB1, CCL5 neutralizing antibodies or normal rabbit IgG. n = 6. Scale bars, 500 μm. (E) Quantification data are shown in ( D) . (F) Basso, Beattie, and Bresnahan (BBB) locomotor scale scores for hindlimb motor function in rats at 0 d, 7 d, 14 d, 21 d and 28 d following the intrathecal administration of the HMGB1 Ab, CCL5 Ab or normal rabbit IgG. n = 6. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Immunofluorescence, Inhibition, Injection, Labeling, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Mechanistic diagram of HMGB1-mediated astrocytic CCL5 contributes to microglia/macrophages activation and recruitment.

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Activation Assay

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors promote T-cell infiltration by upregulating CCL5 chemokine. (a) Chord diagram of gene correlation in the Arf1-ablated MYC-ON liver tumors ( n = 4 in each group). (b) qRT-PCR for chemokines in CT26 cells treated with DMSO, DU101, or DU102. (c) Serum CCL5 levels in the CT26 allografts with the indicated treatments ( n = 6 in each group). (d) CCL5 mRNA levels in the liver tumors of MYC-ON mice that were treated with DMSO, DU101, or DU102 ( n = 9 in each group). (e) Experimental design for the co-culture system. (f) FACS analysis of the migration of CD3 + T cells in CT26 cells that were treated with DMSO, DU101, or DU102. (g) The mRNA levels of CCL5 in CT26 cells with the indicated knockdowns and treatments. (h) FACS analysis of CD3 + T-cell migration in CT26 cells with the indicated knockdowns and treatments. (i and j) Tumor volumes (i) and tumor weights (j) of CT26 allografts with the indicated knockdowns and treatments ( n = 10 in each group). (k) Percentages of infiltrated CD3 + T cells in CT26 allografts with the indicated knockdowns and treatments ( n = 3 in each group). Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Co-Culture Assay, Migration

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors activate the PPARγ pathway by inducing unsaturated fatty acid (LPE). (a) Heatmap of different lipid fractions in the Arf1-deficient CT26 cells. (b) The mRNA levels of downstream genes of PPARγ were detected in CT26 cells treated with different concentrations of LPE (0, 100, 200, 250, and 300 μmol/L for 24 h) by qRT-PCR. (c and d) The mRNA levels of PPARγ in CT26 cells (c) and MYC-ON liver tumors (d) treated with DMSO or Arf1 inhibitors were detected by qRT-PCR. (e) The CCL5 mRNA levels in CT26 cells treated with different concentrations of LPE were detected by qRT-PCR. (f) The CCL5 mRNA levels in CT26 cells with vesicle or GW9662 treatment were detected by qRT-PCR. (g) The CCL5 levels in cell medium collected from CT26 cells with the indicated treatments were determined by ELISA. (h) The percentages of the infiltrated CD3 + T cells in CT26 cells with the indicated treatments. Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors promote T-cell tumor infiltration by activating the NF-κB-CCL5 pathway. (a) The phosphorylated p65 in CT26 cells treated with vesicle, DU101, or DU102 was detected by western blot analysis. (b) The phosphorylated p65 in MYC-ON liver tumors after DMSO, DU101, or DU102 treatment was detected by IHC staining. The right was the statistical analysis of p-p65 + cells per field. (c) The relative CCL5 enrichment in CT26 cells with the indicated treatments was analyzed by ChIP-PCR assay. (d) The CCL5 mRNA levels in CT26 cells with the indicated treatments were detected by qRT-PCR. (e) The CCL5 levels in the cell medium collected from CT26 cells with the indicated treatments were detected by the mouse CCL5 ELISA kit. (f) FACS analysis of the infiltrated CD3 + T cells in CT26 cells with the indicated treatments. (g and h) The proportions of the infiltrated CD3 + T cells (g) and CD3 + CCR5 + T (h) cells in Hepa1-6 cells with the indicated treatments were detected by FACS analysis. Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Western Blot, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Chemistry

Article Title: Activated platelet membrane nanovesicles recruit neutrophils to exert the antitumor efficiency

doi: 10.3389/fchem.2022.955995

Figure Lengend Snippet: Characteristics of PLTm nanovesicles. (A) PLTm nanovesicles observed under TEM, scale bar: 100 nm. (B) TEM size distribution of PLTm nanovesicles. (C) DLS of PLTm nanovesicles. (D) Zeta potential of PLTm nanovesicles. (E) SDS-PAGE protein analysis of PLTs and activated PLTm nanovesicles. M: Marker, P: PLTs, aPNs: activated PLTm nanovesicles. (F) The expression of proteins (P-selectin, ICAM-2 and CCL5) in PLTs and activated PLTm nanovesicles were analyzed by Western blot. aPNs: activated PLTm nanovesicles.

Article Snippet: Bovine serum albumin (BSA) was procured from Yeasen Biological Technology Co., Ltd. BCA Protein Quantitation Kit and Coomassie Blue Fast Staining Solution were from Beyotime Biology Co., Ltd. Polyvinylidene fluoride (PVDF) membrane was produced by Cell Signaling Technology, Co., Ltd. P-selectin rabbit primary antibody was provided by Shanghai Lianshuo Biological Technology Co., Ltd. ICAM-2 (F-5) mouse primary antibody was purchased from Santa Cruz Biotechnology Co., Ltd. CCL5 rabbit primary antibody was provided by Solarbio Biotechnology Co., Ltd. β-Actin (60008-1-Ig) mouse primary antibody was provided by Proteintech Group, Inc. Servicebio Biological Technology Co., Ltd. provided hematoxylin and eosin (HE), Ki-67 immunofluorescence detection kit, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) immunofluorescence detection kit.

Techniques: Zeta Potential Analyzer, SDS Page, Marker, Expressing, Western Blot