Journal: Redox biology

Article Title: Cold atmospheric plasma sensitizes head and neck cancer to chemotherapy and immune checkpoint blockade therapy.

doi: 10.1016/j.redox.2023.102991

Figure Lengend Snippet: Fig. 7. CAPPZ2 reprograms cancer metabolism and weakens oncogenic proliferative signaling in HNC cells. (A) CAPPZ2 treatment reduces protein expression levels of six glycolytic enzymes in Fadu, Cal27, A375, and Hth83 cells. (B) RNA levels of Glut1, HK1, HK2, LDHA, PKM2 and PDK1 decrease at 24 h in response to 60 s of CAPPZ2 treatment in Fadu, Cal27, A375, and Hth83 cells. (C) Lactate production after CAPPZ2-treated 60s in Fadu, Cal27, A375, and Hth83 cells at 24 h. Data expressed as mean ± SD, n = 3, and significance is denoted as ***P < 0.001. (D) These effects are predicted to disrupt glycolysis at multiple enzymatic points, thereby impeding cross-talk for maintaining redox balance in HNC cells. (E) CAPPZ2 reduces protein expression of PI3K and HIF-1α, blocking phosphorylation of mTOR and AKT at 24 h post-treatment in Fadu, Cal27, A375, and Hth83 cells. β-actin was used as a loading control. (F) This facilitates attenuation of the PI3K/AKT/mTOR/HIF-1α signaling pathway at multiple nodes.

Article Snippet: The primary antibodies, including ki67 (Abcam, 1:200), MMP2 (Proteintech, 1:200), MMP9 (Proteintech, 1:200), Ecadherin (Proteintech, 1:1000), N-cadherin (Proteintech, 1:1000), HK1 (Cell Signaling Technology, 1:400), PKM2 (Cell Signaling Technology, 1:200), PDK1 (Cell Signaling Technology, 1:400), LDHA (Cell Signaling Technology, 1:200), CD3 (Proteintech, 1:400), CD4 (Cell Signaling Technology, 1:100), and CD8 (Cell Signaling Technology, 1:100) were incubated with the slides overnight at 4 ◦C.

Techniques: Expressing, Blocking Assay, Phospho-proteomics, Control

Journal: Journal of Translational Medicine

Article Title: HOXA9 drives lymphatic metastasis by activating the c-MYC-glycolysis-lactate axis in gastric cancer

doi: 10.1186/s12967-025-07326-0

Figure Lengend Snippet: HOXA9 overexpression enhances glycolytic metabolism in GC. ( a ) Gene Set Enrichment Analysis (GSEA) plot showing enrichment of glycolysis pathway in HOXA9-high vs. HOXA9-low GC tumors; ( b ) Western blot analysis shows HOXA9 overexpression upregulates glycolysis-related proteins (HIF1α, HK2, GLUT1, PDK1, LDHA) in AGS cells, with β-actin as loading control (representative of three independent experiments); ( c ) Densitometric quantification confirms significant upregulation of glycolytic proteins in HOXA9-overexpressing AGS cells versus vector control ( n = 3, unpaired t-test); ( d ) Western blot analysis demonstrates HOXA9 knockdown downregulates glycolytic proteins in HGC-27 cells (representative of three independent experiments); ( e ) Densitometric quantification shows significant reduction of glycolytic proteins in HOXA9-knockdown HGC-27 cells versus scramble control ( n = 3, unpaired t-test); ( f ) Western blot analysis of subcutaneous xenograft tumors reveals decreased glycolytic protein expression in HOXA9-knockdown group; ( g ) Densitometric quantification confirms significant downregulation of glycolytic proteins in HOXA9-knockdown xenograft tumors ( n = 3 tumors/group, unpaired t-test); ( h ) HOXA9 overexpression promotes glucose uptake in AGS cells, while HOXA9 knockdown reduces uptake in HGC-27 cells ( n = 3, unpaired t-test); ( i ) HOXA9 overexpression increases lactate secretion in AGS cells, while knockdown decreases secretion in HGC-27 cells ( n = 3, unpaired t-test); ( j ) HOXA9 overexpression enhances extracellular acidification rate (ECAR) in AGS cells, while knockdown reduces ECAR in HGC-27 cells ( n = 3, unpaired t-test); ( k ) HOXA9 overexpression suppresses oxygen consumption rate (OCR) in AGS cells, while knockdown increases OCR in HGC-27 cells ( n = 3, unpaired t-test). Data are presented as mean ± SEM

Article Snippet: Membranes were incubated with primary antibodies targeting specific proteins: HOXA9 (Cat. No. ab140631, 1:5000 dilution, Abcam, USA), c-Myc (Cat. No. ab32072, 1:1000 dilution, Abcam, USA), HK2 (Cat. No. 66974-1-Ig, 1:10,000 dilution, Proteintech, USA), HIF-1α (Cat. No. ab16066, 1:2000 dilution, Abcam, USA), GLUT1 (Cat. No. ab115730, 1:100,000 dilution, Abcam, USA), PDK1 (Cat. No. 100261-AP, 1:1000 dilution, Proteintech, USA), and LDHA (Cat. No. 21799-1-AP, 1:10,000 dilution, Proteintech, USA). β-actin (Cat. No. 66009-1-Ig, 1:5000 dilution, Proteintech, USA) was used as the internal loading control to normalize protein loading across samples.

Techniques: Over Expression, Western Blot, Control, Plasmid Preparation, Knockdown, Expressing

Journal: Journal of Translational Medicine

Article Title: HOXA9 drives lymphatic metastasis by activating the c-MYC-glycolysis-lactate axis in gastric cancer

doi: 10.1186/s12967-025-07326-0

Figure Lengend Snippet: HOXA9 drives glycolysis in gastric cancer via c-MYC-dependent mechanisms. ( a ) Representative immunohistochemistry images of HOXA9, c-MYC, and LDHA expression in GC tissues with low and high expression levels. ( b ) Positive correlation between HOXA9 and c-MYC expression in GC tissues ( n = 42, Pearson correlation). ( c ) Positive correlation between HOXA9 and LDHA expression in GC tissues ( n = 42, Pearson correlation). ( d ) Western blot analysis of glycolytic proteins in AGS cells under four conditions: Vector, HOXA9 overexpression (oe-HOXA9), oe-HOXA9 + si-NC, and oe-HOXA9 + si-c-MYC. ( e ) Densitometric quantification of glycolytic proteins (HIF1α, HK2, GLUT1, PDK1, LDHA) showing HOXA9-induced upregulation is attenuated by c-MYC knockdown ( n = 3 independent experiments, one-way ANOVA with Tukey’s test). ( f ) Glucose uptake in AGS cells showing HOXA9-dependent enhancement rescued by c-MYC knockdown ( n = 3, one-way ANOVA with Tukey’s test). ( g ) Lactate secretion in AGS cells showing HOXA9-dependent increase rescued by c-MYC knockdown ( n = 3, one-way ANOVA with Tukey’s test). ( h ) Extracellular acidification rate (ECAR) in AGS cells showing HOXA9-enhanced glycolysis rescued by c-MYC knockdown ( n = 3, one-way ANOVA with Tukey’s test). ( i ) Oxygen consumption rate (OCR) in AGS cells showing HOXA9-suppressed oxidative phosphorylation rescued by c-MYC knockdown ( n = 3, one-way ANOVA with Tukey’s test). Data are presented as mean ± SEM. Exact p-values are shown directly on the graphs

Article Snippet: Membranes were incubated with primary antibodies targeting specific proteins: HOXA9 (Cat. No. ab140631, 1:5000 dilution, Abcam, USA), c-Myc (Cat. No. ab32072, 1:1000 dilution, Abcam, USA), HK2 (Cat. No. 66974-1-Ig, 1:10,000 dilution, Proteintech, USA), HIF-1α (Cat. No. ab16066, 1:2000 dilution, Abcam, USA), GLUT1 (Cat. No. ab115730, 1:100,000 dilution, Abcam, USA), PDK1 (Cat. No. 100261-AP, 1:1000 dilution, Proteintech, USA), and LDHA (Cat. No. 21799-1-AP, 1:10,000 dilution, Proteintech, USA). β-actin (Cat. No. 66009-1-Ig, 1:5000 dilution, Proteintech, USA) was used as the internal loading control to normalize protein loading across samples.

Techniques: Immunohistochemistry, Expressing, Western Blot, Plasmid Preparation, Over Expression, Knockdown, Phospho-proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2 in SGC7901 cells. The expression of GAPDH was used as an internal control. The SGC7901 cells were treated with 0, 2.5 and 5 mg/ml Huaier for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2. The expression of GAPDH was used as an internal control. MKN45 cells were treated with 0, 2.5 and 5 mg/ml for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: IFNλ-dependent activation of ERK/RSK1 and mTOR signaling cascades in HT-29 cells. A–F, serum-starved HT-29 cells were pretreated for 60 min with U0126 or rapamycin and were either left untreated or treated with IFNλ, in the continuous presence or absence of rapamycin or U0126, as indicated. The cells were lysed, and total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of RSK1 on Thr359/Ser363 or against RSK1 (A); with antibodies against the phosphorylated forms of ERK on Thr202/Tyr204 or against ERK (B); with antibodies against the phosphorylated form of eIF4B on Ser422 or against eIF4B (C); with antibodies against the phosphorylated form of mTOR on Ser2448 or against GAPDH (D); with antibodies against the phosphorylated form of p70S6K on Thr421/Ser424 or against p70S6K (E); or with antibodies against the phosphorylated forms of 4E-BP1 on Thr37/46 or against GAPDH (F), as indicated.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Activation Assay, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: IFNλ-dependent activation of RSK1 and mTOR pathways in ARPE-19 cells. A–C, serum-starved ARPE-19 cells were pretreated with rapamycin or U0126 as indicated and then treated with IFNλ for the indicated times. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-Thr359/Ser363 RSK1, anti-RSK1, anti-phospho-Ser422-eIF4B, or anti-eIF4B antibodies (A); with anti-phospho-Thr37/46-4E-BP1 or anti-GAPDH antibodies (B); or with antibodies against the phosphorylated form of p70S6K on Thr421/Ser424 or against p70S6K or anti-GAPDH (C), as indicated.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Activation Assay, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: Binding of 4E-BP1 and RSK1 to the 7-methylguanosine cap complex prevents recruitment of eIF4G, eIF4A, and eIF4E to the cap complex. A, serum-starved HT-29 cells were pretreated for 60 min with U0126 or rapamycin and were either left untreated or treated with IFNλ, in the continuous presence or absence of rapamycin or U0126, as indicated. Cell lysates were bound to the cap analog m7GTP conjugated to beads, and bound proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. B, HT-29 cells were treated as indicated and assayed as described in A. Bound proteins were immunoblotted with the indicated antibodies. C, serum-starved HT-29 cells were pretreated for 6 h with SL0101-1 and were subsequently treated with IFNλ, as indicated. D, serum-starved HT-29 cells were pretreated for 60 min with U0126 or rapamycin and then treated with IFNλ, in the continuous presence or absence of rapamycin or U0126, as indicated. Equal amounts of cell lysates were immunoprecipitated (IP) with an anti-4E-BP1 antibody, and immune complexes were resolved by SDS-PAGE and immunoblotted with anti-RSK1 or anti-4E-BP1 antibodies, as indicated. E, HT-29 cells were pretreated for 6 h with SL0101-1 and were left untreated or treated with IFNλ, in the continuous presence or absence of inhibitor, as indicated. Equal amounts of cell lysates were immunoprecipitated with anti-4EBP1 antibodies, and immune complexes were resolved by SDS-PAGE for analysis of 4E-BP1 and RSK1, as indicated. F, HT-29 cells were transfected with either control siRNA or siRNA specifically targeting 4E-BP1 and treated with IFNλ, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies, against 4E-BP1 or GAPDH, as indicated. G, cells were transfected with either control siRNA or siRNA specifically targeting 4E-BP1 and treated with IFNλ, as indicated. Cell lysates were bound to the cap analog m7GTP conjugated to beads, and bound proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Binding Assay, SDS Page, Immunoprecipitation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: Binding of inactive RSK1 to 4E-BP1. A, equal amounts of GST-RSK1 or GST-RSK1 (activated) were annealed with 4E-BP1-His and then bound to the His affinity column. Equal amounts from fractions collected from the affinity column were resolved by SDS-PAGE and immunoblotted with antibodies against GST or 4E-BP1, as indicated. B, input protein levels from the experiment shown in A for GST-RSK1 (sample A) or GST-RSK1 activated (sample B). Immunoblotting with anti-GST or the anti-phospho-Ser221 RSK1 or anti-4E-BP1 (to detect His-4E-BP1) is shown. C, equal amounts of GST-RSK1 were subjected to in vitro kinase assays using active ERK1 and active PDK1, in the presence or absence of the SL0101-1 inhibitor, and then were annealed with 4E-BP1-His and bound to the His affinity column. After extensive washing, proteins were eluted from the column, and equal amounts from each eluted sample were resolved by SDS-PAGE and immunoblotted with antibodies against GST and 4E-BP1, as indicated. D, equal amounts of GST-RSK1 (activated) or GST-RSK1 (activated) that was subjected to an in vitro phosphatase (PP2A) assay were annealed with 4E-BP1-His. After binding to the His affinity column and extensive washing, proteins were eluted from the column, and equal amounts of eluted samples were resolved by SDS-PAGE and immunoblotted with antibodies against GST or 4E-BP1, as indicated. E, input protein levels from the experiment shown in D (panel A) for activated GST-RSK1 (sample A) or activated GST-RSK1 after subjected to in vitro phosphatase (PP2A) assay (sample B) or GST (sample C). Immunoblotting with anti-GST, anti-phospho-Ser221 RSK1 or anti-4E-BP1 (to detect 4E-BP1-His) is shown.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Binding Assay, Affinity Column, SDS Page, Western Blot, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: RSK1 activity is required for IFNλ-dependent phosphorylation of 4E-BP1 on Thr37/46. A, serum-starved HT-29 cells were pretreated with SL0101-1 or diluent for 6 h and then treated with IFNλ for the indicated times, in the continuous presence or absence of SL0101-1, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Equal cell lysates from the same experiment were analyzed separately by SDS-PAGE and immunoblotted with an anti-phospho-RSK1 (Ser221) and anti-RSK1. B, HT29 cells were transfected with either control siRNA or siRNA targeting RSK1 and treated with IFNλ, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, serum-starved HT-29 cells were pretreated with U0126 for 1 h and then treated with IFNλ for 90 min. The cells were lysed, and equal amounts of protein were immunoprecipitated (IP) with an anti-RSK1 antibody. In vitro kinase assays to detect RSK activity were subsequently carried out on the immunoprecipitates, using a 4E-BP1-His protein as an exogenous substrate. D, serum-starved HT-29 cells were pretreated with SL0101-1 for 6 h or BI-D1870 for 1 h and then treated with IFNλ for the indicated times. The cells were lysed, and equal amounts of protein were immunoprecipitated with an anti-RSK1 antibody. In vitro kinase assays to detect RSK activity were subsequently carried out on the immunoprecipitates, using a GST-4E-BP1 protein as an exogenous substrate.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Activity Assay, SDS Page, Transfection, Immunoprecipitation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFN? Signaling *

doi: 10.1074/jbc.M110.183566

Figure Lengend Snippet: RSK1 associates with 4E-BP1 in the 7-methylguanosine cap complex. A, serum-starved 4E-BP1+/+ and 4E-BP1−/− cells were treated with mouse IFNα for the indicated times. Equal amounts of cell lysates were incubated with cap analog beads, and after intensive washing, the retained proteins were resolved by SDS-PAGE and immunoblotted with antibodies against RSK1, 4E-BP1, or eIF4E. B, total cell lysates from the same experiment shown in A were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Article Snippet: SL0101-1 was used at a final concentration of 80 μ m , and BI-D1870 was used at a final concentration of 10 μ m . GST-4E-BP1, His-tagged 4E-BP1, and active PDK1 were from SignalChem (Richmond, Canada).

Techniques: Incubation, SDS Page