Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: ENO1 is upregulated in fibrotic lungs from human and bleomycin-treated mice. (A, B) IHC staining of ENO1 was performed in commercially available human normal (n = 3) and fibrosis (n = 3) lung FFPE sections. (C, D) After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 7) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of FFPE sections and subjected to IHC staining of ENO1. Representative pictures (A, C) and quantitative results of ENO1-stained positive areas were shown (B, D) . After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 4) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of lysates and subjected to Western blotting for ENO1 (E) and the relative densitometry was shown below the representative blot after GAPDH normalization. Cropped blots were shown, and supplementary Fig. presented the full-length blots. Quantitative results were shown by fold change after bleomycin treatment (F) . Scale bar, 100 µM. * P < 0.05, ** P < 0.01. (A, B) One experiment was performed and each picture or data point was from one human subject. (C-F) Data were representative for two independent experiments. Each picture, data point, or protein band was from one mouse except ( F ) was shown as mean ± SD

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Immunohistochemistry, Injection, Staining, Western Blot

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 ameliorates lung fibrosis in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. Mouse lungs were harvested on day 14 or 21 for preparation of FFPE sections and subjected to H&E staining (A) and Masson’s trichrome staining (B) . Representative pictures on day 21 (A, B) and quantitative results of Ashcroft score on day 21 (C) and inflammation score on day 14 (D) were shown. Body weight change (E) , ratio of lung weight versus body weight (F) , collagen content of the lungs (G) , and levels of TGF-β in BALF (H) were shown. Scale bar, 100 µM. * P < 0.05, ** P < 0.01, *** P < 0.001. Data were representative for two independent experiments. Each picture or data point was from one mouse except (E) was shown as mean ± SEM

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Blocking Assay, Injection, Staining

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduced PBMC migration and immune cells recruitment to the alveolar space and lung interstitium in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. (A) Pooled PBMCs of each group (n = 5) were collected on day 7 & 14 and subjected to migration assay. BALF (B) was collected from the groups of sham (n = 6), BLM + Vehicle (n = 10), and BLM + HL217 (n = 9) on day 4, which was then assessed by using flow cytometry for the number total cell, neutrophil (CD11c − /Ly6G + /Ly6B.2 + cells), or monocyte (CD11c − /Ly6G − /Ly6B.2 + cells). The perfused lungs (C, D) were collected from the groups of sham (n = 6), BLM + Vehicle (n = 6), and BLM + HL217 (n = 5) on day 7. The collected cell samples were subjected to flow cytometry analysis for tissue-resident alveolar macrophages (TR-AM) (CD45 + CD11c + SigF + ), neutrophils (CD45 + CD11b + Gr-1 + ), eosinophils (CD45 + CD11c − SigF + ), constitutive monocytes/macrophages (Gr-1 − MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 − ), classical MoMp (Gr-1 + MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 + ), dendritic cells (DC) (CD45 + CD11b + MHC-II + CD64 − CD24 + ), monocyte-derived alveolar macrophage (Mo-AM) (CD45 + CD11b + MHC-II + CD64 + CD11c + ), and interstitial macrophages (IM) (CD45 + CD11b + MHC-II + CD64 + CD11c − ). * P < 0.05, ** P < 0.01, *** P < 0.001. (A, C, D) Data were representative for two independent experiments. (B) Data were pooled from two independent experiments. Each data point was from one mouse except (A) was shown as fold change and each group contained three technical replicates of pooled blood from five mice

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Blocking Assay, Migration, Injection, Flow Cytometry, Derivative Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in LPS-stimulated primary human PBMC. Fresh peripheral blood was collected from 3 healthy donors and added with indicated concentrations of LPS, control human IgG1 (hIgG1), and HL217 for 4 h, followed by isolation of PBMC. The isolated PBMC was subjected to measurement of cell-associated plasmin activity (A) and cell migration (B) . (C-F) PBMC was isolated from 3 healthy donors and treated with indicated concentrations of LPS, hIgG1, and HL217 for 24 h. Cell culture supernatants were collected and subjected to measurement of pro-inflammatory cytokines, including TNF-α (C) , IL-1β (D) , IL-6 (E) , and CCL2 (F) , by ELISA. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the LPS-treated group. Pooled data of three independent experiments and each data point was from one human subject

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Isolation, Activity Assay, Migration, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in bleomycin-stimulated primary human endothelial cells (HUVEC). Primary human umbilical vascular endothelial cells (HUVEC) were treated with 50 ng/ml bleomycin (BLM) for 24 h and subjected to the measurement of surface ENO1 expression (A, B) by using immunofluorescence staining. (A) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green). The lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (B) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. (C-E) HUVEC were treated with indicated concentrations of BLM, HL217, hIgG1, and plasmin inhibitor tranexamic acid (TXA) for 24 h. Cell-associated plasmin activity was measured (C) and cell culture supernatants were collected for measurement of chemokines, including CCL2 (D) and IL-8 (E) , by ELISA. Scale bar, 100 µM. # P < 0.05, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the BLM-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group)

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Expressing, Immunofluorescence, Staining, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-fibrotic effects of ENO1 Ab HL217 in TGF-β-stimulated primary human lung fibroblasts. Primary normal human lung fibroblasts (NHLF) (A, C) or diseased human lung fibroblasts from IPF patient (DHLF-IPF) (B, D) were treated with 10 ng/ml TGF-β for 24 h and subjected to the measurement of surface ENO1 expression by using immunofluorescence staining. (A, B) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green) and lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (C, D) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. NHLF (E, G) and DHLF-IPF (F, H) were treated with 10 ng/ml TGF-β for 4 h and allowed to migrate for another 18 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA in the migration assay (E, F) or the CXCL12 chemotaxis assay (G, H) . # P < 0.05, ## P < 0.01 vs. untreated group; * P < 0.05, ** P < 0.01 vs. TGF-β-treated group ( E , F ) or TGF-β- and CXCL12-treated group (G, H); & P < 0.05 vs. the group with only CXCL12 as chemoattractant. Pooled data of two independent experiments were shown as mean ± SD (one technical replicate and one biological repeat per group for each experiment)

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Expressing, Immunofluorescence, Staining, Migration, Chemotaxis Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduces plasmin activation and collagen secretion in primary human lung fibroblasts. (A) DHLF-IPF was allowed to grow for 48 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. Untreated NHLF was cultured as control. The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. (B) Primary normal human lung myofibroblasts (NHLF-Myo) were differentiated from NHLF by treatment with TGF-β for 72 h. Increasing expression of fibronectin and α-SMA confirmed the differentiation of NHLF-Myo. Cropped blots were shown, and supplementary Fig. S7 presented the full-length blots. (C) The NHLF-Myo was treated with TGF-β for another 4 h and subjected to the measurement of cell-associated plasmin activity in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. (D) The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. # P < 0.05, ### P < 0.001 vs. the untreated group; && P < 0.01 vs. the NHLF group; * P < 0.05, ** P < 0.01 vs. the TGF-β-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group) except ( B ) was representative for two independent experiments (each protein band was from one biological repeat)

Article Snippet: To stain surface ENO1, cells grown on poly-L-lysine-coated 15 mm coverslips were fixed in 4% paraformaldehyde (#47317, Alfa Aesar) for 15 min at 4 o C, blocked in blocking buffer for 1 h at room temperature, and incubated with anti-ENO1 primary antibody (#ab190365, Abcam, 1:250) for overnight at 4 o C. Since HL217 was developed for therapeutic purpose and not tested for application of IF staining, to ensure the reliability of the results, we used the commercially available anti-ENO1 antibody because it is suitable for IF staining as tested by the supplier.

Techniques: Blocking Assay, Activation Assay, Cell Culture, Expressing, Activity Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: ENO1 is upregulated in fibrotic lungs from human and bleomycin-treated mice. (A, B) IHC staining of ENO1 was performed in commercially available human normal (n = 3) and fibrosis (n = 3) lung FFPE sections. (C, D) After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 7) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of FFPE sections and subjected to IHC staining of ENO1. Representative pictures (A, C) and quantitative results of ENO1-stained positive areas were shown (B, D) . After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 4) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of lysates and subjected to Western blotting for ENO1 (E) and the relative densitometry was shown below the representative blot after GAPDH normalization. Cropped blots were shown, and supplementary Fig. presented the full-length blots. Quantitative results were shown by fold change after bleomycin treatment (F) . Scale bar, 100 µM. * P < 0.05, ** P < 0.01. (A, B) One experiment was performed and each picture or data point was from one human subject. (C-F) Data were representative for two independent experiments. Each picture, data point, or protein band was from one mouse except ( F ) was shown as mean ± SD

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Immunohistochemistry, Injection, Staining, Western Blot

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 ameliorates lung fibrosis in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. Mouse lungs were harvested on day 14 or 21 for preparation of FFPE sections and subjected to H&E staining (A) and Masson’s trichrome staining (B) . Representative pictures on day 21 (A, B) and quantitative results of Ashcroft score on day 21 (C) and inflammation score on day 14 (D) were shown. Body weight change (E) , ratio of lung weight versus body weight (F) , collagen content of the lungs (G) , and levels of TGF-β in BALF (H) were shown. Scale bar, 100 µM. * P < 0.05, ** P < 0.01, *** P < 0.001. Data were representative for two independent experiments. Each picture or data point was from one mouse except (E) was shown as mean ± SEM

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Blocking Assay, Injection, Staining

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduced PBMC migration and immune cells recruitment to the alveolar space and lung interstitium in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. (A) Pooled PBMCs of each group (n = 5) were collected on day 7 & 14 and subjected to migration assay. BALF (B) was collected from the groups of sham (n = 6), BLM + Vehicle (n = 10), and BLM + HL217 (n = 9) on day 4, which was then assessed by using flow cytometry for the number total cell, neutrophil (CD11c − /Ly6G + /Ly6B.2 + cells), or monocyte (CD11c − /Ly6G − /Ly6B.2 + cells). The perfused lungs (C, D) were collected from the groups of sham (n = 6), BLM + Vehicle (n = 6), and BLM + HL217 (n = 5) on day 7. The collected cell samples were subjected to flow cytometry analysis for tissue-resident alveolar macrophages (TR-AM) (CD45 + CD11c + SigF + ), neutrophils (CD45 + CD11b + Gr-1 + ), eosinophils (CD45 + CD11c − SigF + ), constitutive monocytes/macrophages (Gr-1 − MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 − ), classical MoMp (Gr-1 + MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 + ), dendritic cells (DC) (CD45 + CD11b + MHC-II + CD64 − CD24 + ), monocyte-derived alveolar macrophage (Mo-AM) (CD45 + CD11b + MHC-II + CD64 + CD11c + ), and interstitial macrophages (IM) (CD45 + CD11b + MHC-II + CD64 + CD11c − ). * P < 0.05, ** P < 0.01, *** P < 0.001. (A, C, D) Data were representative for two independent experiments. (B) Data were pooled from two independent experiments. Each data point was from one mouse except (A) was shown as fold change and each group contained three technical replicates of pooled blood from five mice

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Blocking Assay, Migration, Injection, Flow Cytometry, Derivative Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in LPS-stimulated primary human PBMC. Fresh peripheral blood was collected from 3 healthy donors and added with indicated concentrations of LPS, control human IgG1 (hIgG1), and HL217 for 4 h, followed by isolation of PBMC. The isolated PBMC was subjected to measurement of cell-associated plasmin activity (A) and cell migration (B) . (C-F) PBMC was isolated from 3 healthy donors and treated with indicated concentrations of LPS, hIgG1, and HL217 for 24 h. Cell culture supernatants were collected and subjected to measurement of pro-inflammatory cytokines, including TNF-α (C) , IL-1β (D) , IL-6 (E) , and CCL2 (F) , by ELISA. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the LPS-treated group. Pooled data of three independent experiments and each data point was from one human subject

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Isolation, Activity Assay, Migration, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in bleomycin-stimulated primary human endothelial cells (HUVEC). Primary human umbilical vascular endothelial cells (HUVEC) were treated with 50 ng/ml bleomycin (BLM) for 24 h and subjected to the measurement of surface ENO1 expression (A, B) by using immunofluorescence staining. (A) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green). The lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (B) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. (C-E) HUVEC were treated with indicated concentrations of BLM, HL217, hIgG1, and plasmin inhibitor tranexamic acid (TXA) for 24 h. Cell-associated plasmin activity was measured (C) and cell culture supernatants were collected for measurement of chemokines, including CCL2 (D) and IL-8 (E) , by ELISA. Scale bar, 100 µM. # P < 0.05, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the BLM-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group)

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Expressing, Immunofluorescence, Staining, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-fibrotic effects of ENO1 Ab HL217 in TGF-β-stimulated primary human lung fibroblasts. Primary normal human lung fibroblasts (NHLF) (A, C) or diseased human lung fibroblasts from IPF patient (DHLF-IPF) (B, D) were treated with 10 ng/ml TGF-β for 24 h and subjected to the measurement of surface ENO1 expression by using immunofluorescence staining. (A, B) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green) and lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (C, D) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. NHLF (E, G) and DHLF-IPF (F, H) were treated with 10 ng/ml TGF-β for 4 h and allowed to migrate for another 18 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA in the migration assay (E, F) or the CXCL12 chemotaxis assay (G, H) . # P < 0.05, ## P < 0.01 vs. untreated group; * P < 0.05, ** P < 0.01 vs. TGF-β-treated group ( E , F ) or TGF-β- and CXCL12-treated group (G, H); & P < 0.05 vs. the group with only CXCL12 as chemoattractant. Pooled data of two independent experiments were shown as mean ± SD (one technical replicate and one biological repeat per group for each experiment)

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Expressing, Immunofluorescence, Staining, Migration, Chemotaxis Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduces plasmin activation and collagen secretion in primary human lung fibroblasts. (A) DHLF-IPF was allowed to grow for 48 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. Untreated NHLF was cultured as control. The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. (B) Primary normal human lung myofibroblasts (NHLF-Myo) were differentiated from NHLF by treatment with TGF-β for 72 h. Increasing expression of fibronectin and α-SMA confirmed the differentiation of NHLF-Myo. Cropped blots were shown, and supplementary Fig. S7 presented the full-length blots. (C) The NHLF-Myo was treated with TGF-β for another 4 h and subjected to the measurement of cell-associated plasmin activity in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. (D) The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. # P < 0.05, ### P < 0.001 vs. the untreated group; && P < 0.01 vs. the NHLF group; * P < 0.05, ** P < 0.01 vs. the TGF-β-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group) except ( B ) was representative for two independent experiments (each protein band was from one biological repeat)

Article Snippet: Endogenous peroxidase activity was quenched followed by blocking and then incubation with anti-ENO1 antibody (#ab227978, Abcam, 1:500) or isotype control (#ab172730, Abcam, 1:500) at 4 o C for overnight.

Techniques: Blocking Assay, Activation Assay, Cell Culture, Expressing, Activity Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: ENO1 is upregulated in fibrotic lungs from human and bleomycin-treated mice. (A, B) IHC staining of ENO1 was performed in commercially available human normal (n = 3) and fibrosis (n = 3) lung FFPE sections. (C, D) After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 7) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of FFPE sections and subjected to IHC staining of ENO1. Representative pictures (A, C) and quantitative results of ENO1-stained positive areas were shown (B, D) . After intratracheal injection of bleomycin (BLM, 3 mg/kg) (n = 4) or PBS vehicle control (Sham) (n = 4), the mouse lungs were harvested on day 21 for preparation of lysates and subjected to Western blotting for ENO1 (E) and the relative densitometry was shown below the representative blot after GAPDH normalization. Cropped blots were shown, and supplementary Fig. presented the full-length blots. Quantitative results were shown by fold change after bleomycin treatment (F) . Scale bar, 100 µM. * P < 0.05, ** P < 0.01. (A, B) One experiment was performed and each picture or data point was from one human subject. (C-F) Data were representative for two independent experiments. Each picture, data point, or protein band was from one mouse except ( F ) was shown as mean ± SD

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Immunohistochemistry, Injection, Staining, Western Blot

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 ameliorates lung fibrosis in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. Mouse lungs were harvested on day 14 or 21 for preparation of FFPE sections and subjected to H&E staining (A) and Masson’s trichrome staining (B) . Representative pictures on day 21 (A, B) and quantitative results of Ashcroft score on day 21 (C) and inflammation score on day 14 (D) were shown. Body weight change (E) , ratio of lung weight versus body weight (F) , collagen content of the lungs (G) , and levels of TGF-β in BALF (H) were shown. Scale bar, 100 µM. * P < 0.05, ** P < 0.01, *** P < 0.001. Data were representative for two independent experiments. Each picture or data point was from one mouse except (E) was shown as mean ± SEM

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Blocking Assay, Injection, Staining

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduced PBMC migration and immune cells recruitment to the alveolar space and lung interstitium in bleomycin-treated mice. After intratracheal injection of 3 mg/kg bleomycin (BLM) (day 0), mice were treated with ENO1 Ab HL217 (10 mg/kg) intravenously on a 6-day interval from day 1. (A) Pooled PBMCs of each group (n = 5) were collected on day 7 & 14 and subjected to migration assay. BALF (B) was collected from the groups of sham (n = 6), BLM + Vehicle (n = 10), and BLM + HL217 (n = 9) on day 4, which was then assessed by using flow cytometry for the number total cell, neutrophil (CD11c − /Ly6G + /Ly6B.2 + cells), or monocyte (CD11c − /Ly6G − /Ly6B.2 + cells). The perfused lungs (C, D) were collected from the groups of sham (n = 6), BLM + Vehicle (n = 6), and BLM + HL217 (n = 5) on day 7. The collected cell samples were subjected to flow cytometry analysis for tissue-resident alveolar macrophages (TR-AM) (CD45 + CD11c + SigF + ), neutrophils (CD45 + CD11b + Gr-1 + ), eosinophils (CD45 + CD11c − SigF + ), constitutive monocytes/macrophages (Gr-1 − MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 − ), classical MoMp (Gr-1 + MoMp) (CD45 + CD11b + MHC-II − CD64 + Gr-1 + ), dendritic cells (DC) (CD45 + CD11b + MHC-II + CD64 − CD24 + ), monocyte-derived alveolar macrophage (Mo-AM) (CD45 + CD11b + MHC-II + CD64 + CD11c + ), and interstitial macrophages (IM) (CD45 + CD11b + MHC-II + CD64 + CD11c − ). * P < 0.05, ** P < 0.01, *** P < 0.001. (A, C, D) Data were representative for two independent experiments. (B) Data were pooled from two independent experiments. Each data point was from one mouse except (A) was shown as fold change and each group contained three technical replicates of pooled blood from five mice

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Blocking Assay, Migration, Injection, Flow Cytometry, Derivative Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in LPS-stimulated primary human PBMC. Fresh peripheral blood was collected from 3 healthy donors and added with indicated concentrations of LPS, control human IgG1 (hIgG1), and HL217 for 4 h, followed by isolation of PBMC. The isolated PBMC was subjected to measurement of cell-associated plasmin activity (A) and cell migration (B) . (C-F) PBMC was isolated from 3 healthy donors and treated with indicated concentrations of LPS, hIgG1, and HL217 for 24 h. Cell culture supernatants were collected and subjected to measurement of pro-inflammatory cytokines, including TNF-α (C) , IL-1β (D) , IL-6 (E) , and CCL2 (F) , by ELISA. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the LPS-treated group. Pooled data of three independent experiments and each data point was from one human subject

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Isolation, Activity Assay, Migration, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-inflammatory effects of ENO1 Ab HL217 in bleomycin-stimulated primary human endothelial cells (HUVEC). Primary human umbilical vascular endothelial cells (HUVEC) were treated with 50 ng/ml bleomycin (BLM) for 24 h and subjected to the measurement of surface ENO1 expression (A, B) by using immunofluorescence staining. (A) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green). The lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (B) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. (C-E) HUVEC were treated with indicated concentrations of BLM, HL217, hIgG1, and plasmin inhibitor tranexamic acid (TXA) for 24 h. Cell-associated plasmin activity was measured (C) and cell culture supernatants were collected for measurement of chemokines, including CCL2 (D) and IL-8 (E) , by ELISA. Scale bar, 100 µM. # P < 0.05, ### P < 0.001 vs. untreated cells; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the BLM-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group)

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Expressing, Immunofluorescence, Staining, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Anti-fibrotic effects of ENO1 Ab HL217 in TGF-β-stimulated primary human lung fibroblasts. Primary normal human lung fibroblasts (NHLF) (A, C) or diseased human lung fibroblasts from IPF patient (DHLF-IPF) (B, D) were treated with 10 ng/ml TGF-β for 24 h and subjected to the measurement of surface ENO1 expression by using immunofluorescence staining. (A, B) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green) and lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (C, D) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. NHLF (E, G) and DHLF-IPF (F, H) were treated with 10 ng/ml TGF-β for 4 h and allowed to migrate for another 18 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA in the migration assay (E, F) or the CXCL12 chemotaxis assay (G, H) . # P < 0.05, ## P < 0.01 vs. untreated group; * P < 0.05, ** P < 0.01 vs. TGF-β-treated group ( E , F ) or TGF-β- and CXCL12-treated group (G, H); & P < 0.05 vs. the group with only CXCL12 as chemoattractant. Pooled data of two independent experiments were shown as mean ± SD (one technical replicate and one biological repeat per group for each experiment)

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Expressing, Immunofluorescence, Staining, Migration, Chemotaxis Assay

Journal: Respiratory Research

Article Title: Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

doi: 10.1186/s12931-023-02583-3

Figure Lengend Snippet: Blocking ENO1 reduces plasmin activation and collagen secretion in primary human lung fibroblasts. (A) DHLF-IPF was allowed to grow for 48 h in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. Untreated NHLF was cultured as control. The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. (B) Primary normal human lung myofibroblasts (NHLF-Myo) were differentiated from NHLF by treatment with TGF-β for 72 h. Increasing expression of fibronectin and α-SMA confirmed the differentiation of NHLF-Myo. Cropped blots were shown, and supplementary Fig. S7 presented the full-length blots. (C) The NHLF-Myo was treated with TGF-β for another 4 h and subjected to the measurement of cell-associated plasmin activity in the absence or presence of indicated concentrations of HL217, hIgG1, or TXA. (D) The cell culture supernatants were collected and subjected to the measurement of collagen by using Sircol assay. # P < 0.05, ### P < 0.001 vs. the untreated group; && P < 0.01 vs. the NHLF group; * P < 0.05, ** P < 0.01 vs. the TGF-β-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group) except ( B ) was representative for two independent experiments (each protein band was from one biological repeat)

Article Snippet: Primary antibodies were purchased from Abcam and listed as antigen (Cat#): ENO1 (#ab5694), fibronectin (#ab2413), α-SMA (#ab5694), and GAPDH (#ab181602).

Techniques: Blocking Assay, Activation Assay, Cell Culture, Expressing, Activity Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 expression in MM tumors is elevated compared with normal bone marrow tissues. A human MM tissue microarray (10 MM cases and 11 normal bone marrow cases, with duplicate cores per case) was used for immunohistochemical staining of ENO1. (A) Representative images were shown at ×10 (upper panels) or ×40 (lower panels) magnification. (B) Quantification of the ENO1-positively stained area. Each dot represents the result from one tissue core. The P-value was calculated with a two-tailed unpaired Student's t-test. ENO1, enolase-1; MM, multiple myeloma.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 knockdown attenuates lactate production, cell migration, cell viability and surface ENO1 expression. RPMI-8226 and U266 cells were transfected with ENO1-targeting siRNA (si-ENO1 #1 or #2) or control siRNA (scramble sequence) for 72 h, and ENO1 depletion efficiency was confirmed by (A and G) western blotting. GAPDH served as the loading control. (B) ENO1-knockdown RPMI 8226 cells were cultured for an additional 48 h, and then the supernatant was collected for determination of lactate levels. (C) Transwell migration assay, (D and H) cell viability assays, and measurement of cell surface ENO1 by (E and I) flow cytometry and (F) an antibody labeling assay were performed. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; siRNA, small interfering RNA.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Migration, Expressing, Transfection, Sequencing, Western Blot, Cell Culture, Transwell Migration Assay, Flow Cytometry, Antibody Labeling, Small Interfering RNA

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Extracellular ENO1 enhances glycolysis and pro-cancer activities. RPMI-8226 cells were treated with the indicated concentrations of recombinant ENO1-WT. The studies were conducted in the presence or absence of 100 µg/ml ENO1 mAb (also termed HuL001). (A) The lactate concentration in the culture medium (upper panel) and intracellular LDH activity (lower panel) were measured 48 h after ENO1-WT treatment. (B) The HIF1A, HK2, PFKFB3, GLUT1 and ENO1 mRNA levels were quantified by reverse transcription-quantitative PCR after 6 h of ENO1-WT treatment. (C) The HIF-1α, HK2, PFKFB3, GLUT1 and ENO1 protein levels were analyzed by immunoblotting after 24 h of ENO1-WT treatment. The amounts of studied proteins were first normalized with GAPDH, and the Rel was then calculated by comparing with the levels in the untreated cells, of which the value is set to 1.0. (D) Cell viability was measured using Cell Counting Kit-8. (E) Secretion of VEGF was measured by ELISA after 48 h of ENO1-WT treatment. (F) The enolase activity of ENO1-WT and two catalytically dead mutants, ENO1-S40A and ENO1-D245R, was measured. RPMI-8226 cells were treated with the indicated concentrations of ENO1-WT, ENO1-S40A and ENO1-D245R for 48 h. The (G) lactate and (H) VEGF concentrations in the culture medium were measured. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; ENO1-WT, wild-type ENO1; GLUT1, glucose transporter 1; HIF1A or HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; LDH, lactate dehydrogenase; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3; Rel., relative ratio; Ut, untreated; VEGF, vascular endothelial growth factor.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Recombinant, Concentration Assay, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Extracellular ENO1 enhances glycolysis and pro-cancer activities through HIF-1α. RPMI-8226 cells were transfected with HIF-1α-targeting siRNA (si-HIF1A) or control siRNA (scramble sequence) for 96 h. (A) The HIF1A, HK2 and GLUT1 mRNA levels were quantified by reverse transcription-quantitative PCR after 6 h of treatment with or without 100 µg/ml ENO1-WT. (B) The HIF-1α, HK2, GLUT1 and IκBα protein levels and the (C) lactate, (D) IL-6 and (E) VEGF concentrations in the culture medium were measured 24 h after treatment with ENO1-WT. The amounts of studied proteins were first normalized with GAPDH, and then the Rel. was calculated by comparing with the scramble siRNA-treated cells, of which the value is set to 1.0. (F) Cell viability and (G) Transwell migration assays of RPMI-8226 cells with or without ENO1-WT treatment were performed following transfection with si-HIF1A or control siRNA. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; ENO1-WT, wild-type ENO1; GLUT1, glucose transporter 1; HIF1A or HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; IL-6, interleukin 6; LDH, lactate dehydrogenase; Rel., relative ratio; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Transfection, Sequencing, Real-time Polymerase Chain Reaction, Migration, Small Interfering RNA

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 mAb reduces glycolytic and pro-cancer activities. (A) The lactate concentrations in the culture medium, collected from ENO1 mAb-treated RPMI-8226 (left panel) and U266 (right panel) cells, were measured after 48 h of ENO1 mAb treatment. In all studies, hIgG1 at the indicated concentrations was included as a specificity control for ENO1 mAb. RPMI-8226 and U266 cells were treated with 100 µg/ml ENO1 mAb or 100 µg/ml hIgG1 for 48 h. (B) Enolase activity and (C) glucose uptake in lysates were further analyzed. Phloretin (a GLUT1 inhibitor) was added at 100 µM as a positive control to inhibit glucose uptake. (D) RPMI-8226 cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 for 24 h. The HIF-1α, HK2, PFKFB3, GLUT1 and ENO1 protein levels were analyzed by immunoblotting. The amounts of studied proteins were first normalized with GAPDH, and then the Rel. was calculated by comparing with the untreated cells, of which the value was set to 1.0. (E) RPMI-8226 (upper panel) or U266 (lower panel) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1. Cell viability was assessed 1, 2 or 3 days after treatment using the Cell Counting Kit-8. The OD 450 nm value was positively associated with the number of viable cells. (F) RPMI-8226 cells were treated with the indicated concentrations of ENO1 mAb or hIgG1. Cell migration was measured by Transwell assay after 18 h of treatment. (G) RPMI-8226 (upper panel) and U266 (lower panel) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 followed by measurement of the plasminogen receptor activity. (H) RPMI-8226 (left panels) or U266 (right panels) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 for 48 h. Supernatant was collected for determination of human VEGF (upper panel) and TGF-β (lower panel) levels by ELISA. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; GLUT1, glucose transporter 1; HIF-1α, hypoxia-inducible factor 1-α; hIgG1, human IgG1; HK2, hexokinase 2; mAb, monoclonal antibody; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3; Rel., relative ratio; TGF, transforming growth factor; Ut, untreated; VEGF, vascular endothelial growth factor.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Activity Assay, Positive Control, Western Blot, Cell Counting, Migration, Transwell Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 mAb reduces tumor growth and glycolysis in vivo in a RPMI-8226 subcutaneous xenograft model. Male nude mice were subcutaneously implanted with RPMI-8226 cells and randomized when the tumor size reached >100 mm 3 (n=6). ENO1 mAb (30 mg/kg) was intraperitoneally injected twice a week at the indicated time points. (A) Each data point represents the mean volume ± SD from the ENO1 mAb-treated, the withdrawing ENO1 mAb treated or vehicle control groups. Mice were sacrificed on day 35 and (B) representative images of excised tumors and the (C) tumor weight are shown. (D) Sera were collected for measurement of lactate concentration. (E) Mice body measurements were collected at the indicated time points. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; mAb, monoclonal antibody.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: In Vivo, Injection, Concentration Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Schematic diagram summarizing the effects and possible mechanisms of extracellular ENO1 in regulating glycolysis and pro-cancer activities. Extracellular ENO1 may enhance glycolytic activity and glycolysis-related genes, including HK2 and GLUT1, through HIF-1α. Moreover, extracellular ENO1 also promoted HIF-1α-mediated pro-cancer activities, such as cell migration, cell viability and production of tumor-promoting cytokines. Consistently, these effects on cancer progression could be attenuated by the ENO1 antibody or ENO1 siRNA. ENO1, enolase-1; GLUT1, glucose transporter 1; HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; siRNA, small interfering RNA.

Article Snippet: Heat-induced epitope retrieval was performed with 0.02 M Tris-EDTA (pH 9.0) using a microwave for 20 min. Endogenous peroxidase activity was quenched then tissues were stained with primary antibody against ENO1 (1:500; cat. no. ab227978; Abcam) or isotype control antibody (1:500; cat. no. ab172730; Abcam) at 4°C overnight.

Techniques: Activity Assay, Migration, Small Interfering RNA

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 expression in MM tumors is elevated compared with normal bone marrow tissues. A human MM tissue microarray (10 MM cases and 11 normal bone marrow cases, with duplicate cores per case) was used for immunohistochemical staining of ENO1. (A) Representative images were shown at ×10 (upper panels) or ×40 (lower panels) magnification. (B) Quantification of the ENO1-positively stained area. Each dot represents the result from one tissue core. The P-value was calculated with a two-tailed unpaired Student's t-test. ENO1, enolase-1; MM, multiple myeloma.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 knockdown attenuates lactate production, cell migration, cell viability and surface ENO1 expression. RPMI-8226 and U266 cells were transfected with ENO1-targeting siRNA (si-ENO1 #1 or #2) or control siRNA (scramble sequence) for 72 h, and ENO1 depletion efficiency was confirmed by (A and G) western blotting. GAPDH served as the loading control. (B) ENO1-knockdown RPMI 8226 cells were cultured for an additional 48 h, and then the supernatant was collected for determination of lactate levels. (C) Transwell migration assay, (D and H) cell viability assays, and measurement of cell surface ENO1 by (E and I) flow cytometry and (F) an antibody labeling assay were performed. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; siRNA, small interfering RNA.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Migration, Expressing, Transfection, Sequencing, Western Blot, Cell Culture, Transwell Migration Assay, Flow Cytometry, Antibody Labeling, Small Interfering RNA

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Extracellular ENO1 enhances glycolysis and pro-cancer activities. RPMI-8226 cells were treated with the indicated concentrations of recombinant ENO1-WT. The studies were conducted in the presence or absence of 100 µg/ml ENO1 mAb (also termed HuL001). (A) The lactate concentration in the culture medium (upper panel) and intracellular LDH activity (lower panel) were measured 48 h after ENO1-WT treatment. (B) The HIF1A, HK2, PFKFB3, GLUT1 and ENO1 mRNA levels were quantified by reverse transcription-quantitative PCR after 6 h of ENO1-WT treatment. (C) The HIF-1α, HK2, PFKFB3, GLUT1 and ENO1 protein levels were analyzed by immunoblotting after 24 h of ENO1-WT treatment. The amounts of studied proteins were first normalized with GAPDH, and the Rel was then calculated by comparing with the levels in the untreated cells, of which the value is set to 1.0. (D) Cell viability was measured using Cell Counting Kit-8. (E) Secretion of VEGF was measured by ELISA after 48 h of ENO1-WT treatment. (F) The enolase activity of ENO1-WT and two catalytically dead mutants, ENO1-S40A and ENO1-D245R, was measured. RPMI-8226 cells were treated with the indicated concentrations of ENO1-WT, ENO1-S40A and ENO1-D245R for 48 h. The (G) lactate and (H) VEGF concentrations in the culture medium were measured. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; ENO1-WT, wild-type ENO1; GLUT1, glucose transporter 1; HIF1A or HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; LDH, lactate dehydrogenase; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3; Rel., relative ratio; Ut, untreated; VEGF, vascular endothelial growth factor.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Recombinant, Concentration Assay, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Extracellular ENO1 enhances glycolysis and pro-cancer activities through HIF-1α. RPMI-8226 cells were transfected with HIF-1α-targeting siRNA (si-HIF1A) or control siRNA (scramble sequence) for 96 h. (A) The HIF1A, HK2 and GLUT1 mRNA levels were quantified by reverse transcription-quantitative PCR after 6 h of treatment with or without 100 µg/ml ENO1-WT. (B) The HIF-1α, HK2, GLUT1 and IκBα protein levels and the (C) lactate, (D) IL-6 and (E) VEGF concentrations in the culture medium were measured 24 h after treatment with ENO1-WT. The amounts of studied proteins were first normalized with GAPDH, and then the Rel. was calculated by comparing with the scramble siRNA-treated cells, of which the value is set to 1.0. (F) Cell viability and (G) Transwell migration assays of RPMI-8226 cells with or without ENO1-WT treatment were performed following transfection with si-HIF1A or control siRNA. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; ENO1-WT, wild-type ENO1; GLUT1, glucose transporter 1; HIF1A or HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; IL-6, interleukin 6; LDH, lactate dehydrogenase; Rel., relative ratio; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Sequencing, Real-time Polymerase Chain Reaction, Migration, Small Interfering RNA

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 mAb reduces glycolytic and pro-cancer activities. (A) The lactate concentrations in the culture medium, collected from ENO1 mAb-treated RPMI-8226 (left panel) and U266 (right panel) cells, were measured after 48 h of ENO1 mAb treatment. In all studies, hIgG1 at the indicated concentrations was included as a specificity control for ENO1 mAb. RPMI-8226 and U266 cells were treated with 100 µg/ml ENO1 mAb or 100 µg/ml hIgG1 for 48 h. (B) Enolase activity and (C) glucose uptake in lysates were further analyzed. Phloretin (a GLUT1 inhibitor) was added at 100 µM as a positive control to inhibit glucose uptake. (D) RPMI-8226 cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 for 24 h. The HIF-1α, HK2, PFKFB3, GLUT1 and ENO1 protein levels were analyzed by immunoblotting. The amounts of studied proteins were first normalized with GAPDH, and then the Rel. was calculated by comparing with the untreated cells, of which the value was set to 1.0. (E) RPMI-8226 (upper panel) or U266 (lower panel) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1. Cell viability was assessed 1, 2 or 3 days after treatment using the Cell Counting Kit-8. The OD 450 nm value was positively associated with the number of viable cells. (F) RPMI-8226 cells were treated with the indicated concentrations of ENO1 mAb or hIgG1. Cell migration was measured by Transwell assay after 18 h of treatment. (G) RPMI-8226 (upper panel) and U266 (lower panel) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 followed by measurement of the plasminogen receptor activity. (H) RPMI-8226 (left panels) or U266 (right panels) cells were treated with the indicated concentrations of ENO1 mAb or hIgG1 for 48 h. Supernatant was collected for determination of human VEGF (upper panel) and TGF-β (lower panel) levels by ELISA. All results are presented as the mean ± SD of three independent experiments. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; GLUT1, glucose transporter 1; HIF-1α, hypoxia-inducible factor 1-α; hIgG1, human IgG1; HK2, hexokinase 2; mAb, monoclonal antibody; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3; Rel., relative ratio; TGF, transforming growth factor; Ut, untreated; VEGF, vascular endothelial growth factor.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Positive Control, Western Blot, Cell Counting, Migration, Transwell Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: ENO1 mAb reduces tumor growth and glycolysis in vivo in a RPMI-8226 subcutaneous xenograft model. Male nude mice were subcutaneously implanted with RPMI-8226 cells and randomized when the tumor size reached >100 mm 3 (n=6). ENO1 mAb (30 mg/kg) was intraperitoneally injected twice a week at the indicated time points. (A) Each data point represents the mean volume ± SD from the ENO1 mAb-treated, the withdrawing ENO1 mAb treated or vehicle control groups. Mice were sacrificed on day 35 and (B) representative images of excised tumors and the (C) tumor weight are shown. (D) Sera were collected for measurement of lactate concentration. (E) Mice body measurements were collected at the indicated time points. P-values were calculated with one-way ANOVA (with Tukey's post hoc test). ENO1, enolase-1; mAb, monoclonal antibody.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: In Vivo, Injection, Concentration Assay

Journal: Oncology Reports

Article Title: Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α

doi: 10.3892/or.2023.8642

Figure Lengend Snippet: Schematic diagram summarizing the effects and possible mechanisms of extracellular ENO1 in regulating glycolysis and pro-cancer activities. Extracellular ENO1 may enhance glycolytic activity and glycolysis-related genes, including HK2 and GLUT1, through HIF-1α. Moreover, extracellular ENO1 also promoted HIF-1α-mediated pro-cancer activities, such as cell migration, cell viability and production of tumor-promoting cytokines. Consistently, these effects on cancer progression could be attenuated by the ENO1 antibody or ENO1 siRNA. ENO1, enolase-1; GLUT1, glucose transporter 1; HIF-1α, hypoxia-inducible factor 1-α; HK2, hexokinase 2; siRNA, small interfering RNA.

Article Snippet: The primary antibodies were as follows: ENO1 (1:2,000; cat. no. ab190365; Abcam), HIF-1α (1:1,000; cat. no. 610958; BD Biosciences), HK2 (1:2,000; cat. no. sc-130358; Santa Cruz Biotechnology, Inc.), 6-phosphofructo-2-kinase/fructose-2,6 biphosphatase 3 (PFKFB3; 1:2,000; cat. no. ab96699; Abcam), GLUT1 (1:5,000; cat. no. ab115730; Abcam), IκBα (1:2,000; cat. no. 9242; Cell Signaling Technology, Inc.), PHD2 (1:1,000; cat. no. sc-271835; Santa Cruz Biotechnology, Inc.) and GAPDH (1:5,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Migration, Small Interfering RNA

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Source, application and concentration of antibodies.

Article Snippet: Rabbit polyclonal HLTF antibody (NBP1-83256) Novus Biologicals (1:100) Rabbit polyclonal CYTB (55090-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal PGAM1 (16126-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal ANXA1 (71–3400) ThermoFisher Scientific (1:20) Rabbit monoclonal γH2AX-phospho S139 (ab81299) Abcam (1:50) Rabbit monoclonal ENO1 (ab227978) Abcam (1:2,000) Rabbit recombinant monoclonal mouse-specific PDPN (MA5-29742) ThermoFisher Scientific (1:500) , IHC-P: Vector Laboratories RTU Biotinylated Goat Anti-rabbit IgG (H+L) (BP-9100-50).

Techniques: Concentration Assay, Recombinant, Plasmid Preparation

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Summary of site-specific S-glutathionylation identified by 2D-DIGE MALDI-TOF/TOF mass spectrometry. Pr-SSG in HLTF -/- CDX/TME.

Article Snippet: Rabbit polyclonal HLTF antibody (NBP1-83256) Novus Biologicals (1:100) Rabbit polyclonal CYTB (55090-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal PGAM1 (16126-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal ANXA1 (71–3400) ThermoFisher Scientific (1:20) Rabbit monoclonal γH2AX-phospho S139 (ab81299) Abcam (1:50) Rabbit monoclonal ENO1 (ab227978) Abcam (1:2,000) Rabbit recombinant monoclonal mouse-specific PDPN (MA5-29742) ThermoFisher Scientific (1:500) , IHC-P: Vector Laboratories RTU Biotinylated Goat Anti-rabbit IgG (H+L) (BP-9100-50).

Techniques: Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: The landscape of immune infiltration between the normal and RA group. (A) Cumulative histogram showing the relative percentages of 22 different immune cell types. (B) Violin plot shows the comparisons between immune cells in normal controls and RA patients. (C) The heatmap shows the correlation in the infiltration of 22 immune cell type proportions. (D-F) Correlation between ENO1, GRN, and PTGS2 with immune infiltrating cells.

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: GWAS analysis to identify the pathogenic regions of the hub genes. The Manhattan plot shows the SNP pathogenic regions corresponding to PTGS2, ENO1 and GRN.

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: GSEA analysis of hub genes. (A) The top 3 most enriched pathways about function enrichment of PTGS2 gene: Nod-like receptor signaling pathway, gene set enriched in FcγR mediated phagocytosis, gene set enriched in the Chemokine signaling pathway. (B) The top 3 most enriched pathways about function enrichment of GRN gene: Lysosome, Leishmania infection, B cell receptor signaling pathway. (C) The top 3 most enriched pathways about function enrichment of ENO1 gene: Lysosome, Pathogenic Escherichia coli infection, Leishmania infection. Screening criteria for significant gene sets included adj. p-value < 0.05 and FDR < 0.5. NES, normalized enrichment score.

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: Enrichment analysis for transcription factors of hub genes (A) Histogram of the AUC. By calculating the AUC, the over-representation of each motif for hub genes was assessed. Red vertical line indicates the degree of significance, whereby motifs with AUC higher than the significance level are considered significant motifs. (B) The recovery curve for the three most significant motifs. Red line represents the global mean of recovered motif curve, green line represents the mean ± standard deviation. Motifs that were larger than the mean ± standard deviation were considered statistically significant. Blue line represents the current motif’s recovered curve. The motif cisbp_M5493 was significantly enriched in hub genes (ENO1 and PTGS2).

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Standard Deviation

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: Single-cell analysis revealed the expression of PTGS2, ENO1 and GRN. (A) Dot plot of hub genes for each cell type. (B) Violin plots showing the expression level of hub genes in nine cell type.

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Single-cell Analysis, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: Validation of the hub genes in experimental samples. (A) Representative tracing of the PI and the joint swelling thickness in rat after CFA or Nor treatment in the two groups(n=12). (B) Representative images of H&E staining of joint sections from AIA rat or Nor rat. (C) The expression of GRN, PTGS2 and ENO1. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. (D) IHC staining for PTGS2, ENO1, and GRN on synovium from samples of OA and RA patients (n = 3).

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Staining, Expressing, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: (A) The expression level of ENO1, ACO1 and ACSL4 in primary FLS of rat model with knockdown of ENO1-siRNA (n=3). (B) The expression level of ENO1 and ACO1 in primary FLS of RA patients(n=3). (C, D) The expression level of ENO1, ACO1 and ACSL4 in primary FLS of rat model and RA patients with knockdown of ENO1-siRNA in the presence of ferrous iron (200 μmol/L), Lip-1 (1 μmol/), Fer-1 (1 μmol/) (n=3). (E) The assessment of cell viability by CCK-8 assay in primary FLS of rat models with knockdown of ENO1-SiRNA in the presence of ferrous iron (200 μmol/L), Lip-1 (1 μmol/), Fer-1 (1 μmol/). Data are means ± SD. *P < 0.05 , ****P < 0.0001 and ns (no significance) versus RA group; ### P < 0.001 versus siRNA group. ****P < 0.0001 versus siRNA group; #P < 0.05 and ## P < 0.01 versus siRNA+Fe2+group. (F) The assessment of cell viability by CCK-8 assay in primary FLS of RA patients with knockdown of ENO1-SiRNA in the presence of ferrous iron (200 μmol/L), Lip-1 (1 μmol/), Fer-1 (1 μmol/). Data are means ± SD. *P < 0.05 , ****P < 0.0001 and ns (no significance) versus RA group; ###P < 0.001 versus siRNA group. *P < 0.05 versus siRNA group; # P < 0.05 versus siRNA+Fe 2+ group.

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Expressing, CCK-8 Assay

Journal: Frontiers in Immunology

Article Title: Identification of potential ferroptosis-associated biomarkers in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1197275

Figure Lengend Snippet: The assessment of lipid peroxidation by flow cytometry in primary FLS of rat model (A) and RA patients (B) with knockdown of ENO1-siRNA in the presence of ferrous iron (200 μmol/L), Lip-1 (1 μmol/), Fer-1 (1 μmol/). (C) The assessment of Intracellular ferrous ion by cellular ferrous ion detection fluorescent probes in primary FLS of rat model and RA patients with knockdown of ENO1-SiRNA in the presence of ferrous iron (200 μmol/L), Lip-1 (1 μmol/), Fer-1 (1 μmol/).

Article Snippet: Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight.

Techniques: Flow Cytometry