Journal: Journal of pharmacological sciences

Article Title: Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.

doi: 10.1016/j.jphs.2019.09.007

Figure Lengend Snippet: Fig. 1. The effects of Ang II on the expression of miR-21, PPARa and fibrosis-related indicators in HK2 cells. (A) and (B) To study the effect of Ang II on miR-21, cells at 60%e70% confluence were growth-arrested in serum-free medium for 24 h and then stimulated with 0, 109 M, 108 M, 107 M, 106 M, or 105 M Ang II for 48 h or 106 M Ang II for 0, 12, 24, 48 or 72 h. The relative miR-21 expression was detected by qRT-PCR and normalized to U6. **P < 0.01, ****P < 0.0001 vs. Control group; ###P < 0.001, ####P < 0.0001 vs. Ang II 106 M group; &&P < 0.01, &&&&P < 0.0001 vs. Ang II 48 h group. (C) and (D) HK2 cells were stimulated with 106 M Ang II for 48 h, and the mRNA and protein levels of PPARa, TGF-b1, Smad3, a-SMA, COL1A1 and FN were determined by qRT-PCR and western blot analyses. p-Smad3 was normalized to t-Smad3. GAPDH was used as an internal reference for the other proteins. (E) Quantitative analysis of the western blot results. The experiments were repeated at least three times. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group; p-Smad3, phospho-Smad3; t-Smad3, total-Smad3.

Article Snippet: HK2 cells in the logarithmic phase were transfected with 50 nM PPARa siRNA (si-PPARa, GenePharma, China) or scramble siRNA (scr siRNA, GenePharma) with Lipofectamine 2000 (Invitrogen, USA) and then stimulated with or without Ang II (MedChemExpress, USA) for 48 h. To investigate the necessity of downregulating PPARa in miR-21-induced lesions, cells transfected with LV-NC or LV-miR-21-OE were treated with pcDNA3.1-PPARa overexpression plasmid (containing human PPARa cDNA, PPARa-OE plasmid, GenePharma) or blank control pcDNA3.1 plasmid (BC plasmid, GenePharma) for 48 h.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Journal of pharmacological sciences

Article Title: Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.

doi: 10.1016/j.jphs.2019.09.007

Figure Lengend Snippet: Fig. 2. miR-21 participated in Ang II-induced fibrosis by activating the TGF-b1/Smad3 pathway, and PPARa was a direct target of miR-21 in HK2 cells. After transfection with LV-NC, LV-miR-21-OE or LV-miR-21-KD, the HK2 cells were treated with or without Ang II for 48 h. (A) The relative miR-21 expression was examined by qRT-PCR and normalized to U6. (B) and (C) The mRNA and protein levels of PPARa, TGF-b1, Smad3, a-SMA, COL1A1 and FN were determined by qRT-PCR and western blot analyses. p-Smad3 was normalized to t-Smad3. GAPDH was used as an internal reference for the other proteins. (D) Quantitative analysis of the western blot results. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. LV- NC group; yP < 0.05, yyP < 0.01, yyyP < 0.001, yyyyP < 0.0001 vs. Ang II þ LV-NC group. (E) Upper part, miR-21 sequence. Lower part, wild-type PPARa 30UTR containing the putative miR-21 binding site and the mutant PPARa 30UTR. (F) HK2 cells were cotransfected with the wild-type or mutant PPARa 30UTR and NC or miR-21 mimics. The dual-luciferase reporter assay confirmed that PPARa is a direct target of miR-21. ***P < 0.001 vs. wild-type PPARa 30UTR þ NC mimics. (G) and (I) Representative immunofluorescence images of TGF-b1 and PPARa at 200 magnification. PPARa, red; TGF-b1, green; DAPI, blue. Scale bars, 50 mm. (H) and (J) Fluorescence intensity of TGF-b1 and PPARa. The experiments were repeated at least three times. The data are presented as the mean ± SD. ***P < 0.001, ****P < 0.0001 vs. LV-NC group; yyyP < 0.001, yyyyP < 0.0001 vs. Ang II þ LV-NC group; p- Smad3, phospho-Smad3; t-Smad3, total-Smad3; WT, wild-type; MUT, mutant.

Article Snippet: HK2 cells in the logarithmic phase were transfected with 50 nM PPARa siRNA (si-PPARa, GenePharma, China) or scramble siRNA (scr siRNA, GenePharma) with Lipofectamine 2000 (Invitrogen, USA) and then stimulated with or without Ang II (MedChemExpress, USA) for 48 h. To investigate the necessity of downregulating PPARa in miR-21-induced lesions, cells transfected with LV-NC or LV-miR-21-OE were treated with pcDNA3.1-PPARa overexpression plasmid (containing human PPARa cDNA, PPARa-OE plasmid, GenePharma) or blank control pcDNA3.1 plasmid (BC plasmid, GenePharma) for 48 h.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Fluorescence

Journal: Journal of pharmacological sciences

Article Title: Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.

doi: 10.1016/j.jphs.2019.09.007

Figure Lengend Snippet: Fig. 3. Downregulation of PPARa promoted renal fibrosis by amplifying the TGF-b1/Smad3 signaling pathway in HK2 cells. HK2 cells were transfected with si-PPARa or scr siRNA with or without Ang II treatment. (A) and (B) The mRNA and protein levels of PPARa, TGF-b1, Smad3, a-SMA, COL1A1 and FN were determined by qRT-PCR and western blot analyses. p-Smad3 was normalized to t-Smad3. GAPDH was used as an internal reference for the other proteins. (C) Quantitative analysis of the western blot results. (D) and (F) Representative immunofluorescence images of PPARa and TGF-b1 at 200 magnification. PPARa, red; TGF-b1, green; DAPI, blue. Scale bars, 50 mm. (E) and (G) Fluorescence in- tensity of PPARa and TGF-b1. The experiments were repeated at least three times. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. scr siRNA group; xP < 0.05, xxP < 0.01, xxxP < 0.001 vs. Ang II þ scr siRNA group; p-Smad3, phospho-Smad3; t-Smad3, total-Smad3.

Article Snippet: HK2 cells in the logarithmic phase were transfected with 50 nM PPARa siRNA (si-PPARa, GenePharma, China) or scramble siRNA (scr siRNA, GenePharma) with Lipofectamine 2000 (Invitrogen, USA) and then stimulated with or without Ang II (MedChemExpress, USA) for 48 h. To investigate the necessity of downregulating PPARa in miR-21-induced lesions, cells transfected with LV-NC or LV-miR-21-OE were treated with pcDNA3.1-PPARa overexpression plasmid (containing human PPARa cDNA, PPARa-OE plasmid, GenePharma) or blank control pcDNA3.1 plasmid (BC plasmid, GenePharma) for 48 h.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Fluorescence

Journal: Journal of pharmacological sciences

Article Title: Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.

doi: 10.1016/j.jphs.2019.09.007

Figure Lengend Snippet: Fig. 4. The miR-21-dependent pathway is the dominant upstream activation signal of the PPARa/TGF-b1/Smad3 pathway in Ang II-treated HK2 cells. HK2 cells were divided into six groups as follows: (1) Control group; (2) Ang II group, HK2 cells were treated with 106 M Ang II for 48 h; (3) Ang II þ LV-NC group, HK2 cells transfected with LV-NC were treated with 106 M Ang II for 48 h; (4) Ang II þ LV-miR-21-KD group, HK2 cells transfected with LV-miR-21-KD were treated with 106 M Ang II for 48 h; (5) Ang II þ Losartan group, HK2 cells were treated with 106 M Ang II and 106 M losartan for 48 h; (6) Ang II þ Losartan þ LV-miR-21-KD group, HK2 cells transfected with LV-miR-21-KD were treated with 106 M Ang II and 106 M losartan for 48 h. (A) The relative miR-21 expression was examined by qRT-PCR and normalized to U6. (B) and (C) The mRNA and protein levels of PPARa, TGF-b1, and Smad3 were determined by qRT-PCR and western blot analyses. p-Smad3 was normalized to t-Smad3. GAPDH was used as an internal reference for the other proteins. (D) Quantitative analysis of the western blot results. The experiments were repeated at least three times. The data are presented as the mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group; yP < 0.05, yyP < 0.01, yyyP < 0.001, yyyyP < 0.0001 vs. Ang II group; p-Smad3, phospho-Smad3; t-Smad3, total-Smad3.

Article Snippet: HK2 cells in the logarithmic phase were transfected with 50 nM PPARa siRNA (si-PPARa, GenePharma, China) or scramble siRNA (scr siRNA, GenePharma) with Lipofectamine 2000 (Invitrogen, USA) and then stimulated with or without Ang II (MedChemExpress, USA) for 48 h. To investigate the necessity of downregulating PPARa in miR-21-induced lesions, cells transfected with LV-NC or LV-miR-21-OE were treated with pcDNA3.1-PPARa overexpression plasmid (containing human PPARa cDNA, PPARa-OE plasmid, GenePharma) or blank control pcDNA3.1 plasmid (BC plasmid, GenePharma) for 48 h.

Techniques: Activation Assay, Control, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of pharmacological sciences

Article Title: Overexpression of microRNA-21 mediates Ang II-induced renal fibrosis by activating the TGF-β1/Smad3 pathway via suppressing PPARα.

doi: 10.1016/j.jphs.2019.09.007

Figure Lengend Snippet: Fig. 5. Low PPARa expression was indispensable for the activation of the TGF-b1/Smad3 pathway and occurrence of renal fibrosis in miR-21-overexpressing cells. HK2 cells transfected with LV-NC or LV-miR-21-OE were further transfected with a BC plasmid or PPARa-OE plasmid. (A) and (B) The mRNA and protein levels of PPARa, TGF-b1, Smad3, a- SMA, COL1A1 and FN were determined by qRT-PCR and western blot analyses. p-Smad3 was normalized to t-Smad3. GAPDH was used as an internal reference for the other proteins. (C) Quantitative analysis of the western blot results. (D) and (F) Representative immunofluorescence images of PPARa and TGF-b1 at 200 magnification. PPARa, red; TGF-b1, green; DAPI, blue. Scale bars, 50 mm. (E) and (G) Fluorescence intensity of PPARa and TGF-b1. The experiments were repeated at least three times. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. LV-NC þ BC group; xP < 0.05, xxP < 0.01, xxxP < 0.001, xxxxP < 0.0001 vs. LV-miR-21-OE þ BC group; BC, blank control plasmid; PPARa OE, PPARa overexpression plasmid; p-Smad3, phospho-Smad3; t-Smad3, total-Smad3.

Article Snippet: HK2 cells in the logarithmic phase were transfected with 50 nM PPARa siRNA (si-PPARa, GenePharma, China) or scramble siRNA (scr siRNA, GenePharma) with Lipofectamine 2000 (Invitrogen, USA) and then stimulated with or without Ang II (MedChemExpress, USA) for 48 h. To investigate the necessity of downregulating PPARa in miR-21-induced lesions, cells transfected with LV-NC or LV-miR-21-OE were treated with pcDNA3.1-PPARa overexpression plasmid (containing human PPARa cDNA, PPARa-OE plasmid, GenePharma) or blank control pcDNA3.1 plasmid (BC plasmid, GenePharma) for 48 h.

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Fluorescence, Control, Over Expression

Journal: BMC cardiovascular disorders

Article Title: β-catenin mediates monocrotaline-induced pulmonary hypertension via glycolysis in rats.

doi: 10.1186/s12872-024-04000-z

Figure Lengend Snippet: Fig. 2 β-catenin enhances glycolysis and NLRP3 inflammasome activation in vitro. A Western blot showing the expression of β-catenin,HK2 and PKM2 in BMDMs. The blots were cropped and hybridized with the corresponding antibodies.Quantitative Western blot results were normalized to those of β-actin. B-D Glucose,PFK and LDH measurements in BMDMs. E Lactate determination in the cell supernatant. F Representative Western blot showing the expression of NLRP3 inflammasome markers in BMDMs from the different groups. β-Actin was used as the internal reference. G-J TNF-α, IL-6, IL-1β and IL-18 levels in the culture supernatants of BMDMs were measured by ELISA. The following comparisons were made: control vs. LPS, LPS vs. LPS + XAV939, LPS vs. LPS + LiCl, and LPS + XAV939 vs. LPS + XAV939 + FBP. The data are presented as the means ± SDs; ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3/group

Article Snippet: The membranes were probed with antibodies against β-catenin (1:1000, Proteintech, Wuhan, China), HK2, PKM2 (1:4000, Proteintech, Wuhan, China), ASC, procaspase-1, caspase-1, NLRP3 (1:1000; all Cell Signaling Technology, USA), β-actin (1:2500, Proteintech, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: BMC cardiovascular disorders

Article Title: β-catenin mediates monocrotaline-induced pulmonary hypertension via glycolysis in rats.

doi: 10.1186/s12872-024-04000-z

Figure Lengend Snippet: Fig. 3 Enhanced glycolysis promotes the activation of the NLRP3 inflammasome in vitro. A The expression of HK2 and PKM2 in BMDMs. β-Actin was used as the internal reference. B-C The levels of the key glycolytic enzymes PFK and LDH were detected in vitro. D Lactate determination in the cell supernatant. E Representative Western blot showing the expression of NLRP3 inflammasome markers in BMDMs from the different groups. The blots were cropped and hybridized with the corresponding antibodies. β-Actin was used as the internal reference. F-I TNF-α, IL-6, IL-1β and IL-18 levels in the culture supernatants of BMDMs were measured by ELISA. The following comparisons were made: control vs. LPS, LPS vs. LPS + 2-DG, and LPS vs. LPS + FBP. The data are presented as the means ± SDs; ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3/group

Article Snippet: The membranes were probed with antibodies against β-catenin (1:1000, Proteintech, Wuhan, China), HK2, PKM2 (1:4000, Proteintech, Wuhan, China), ASC, procaspase-1, caspase-1, NLRP3 (1:1000; all Cell Signaling Technology, USA), β-actin (1:2500, Proteintech, Wuhan, China).

Techniques: Activation Assay, In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: BMC cardiovascular disorders

Article Title: β-catenin mediates monocrotaline-induced pulmonary hypertension via glycolysis in rats.

doi: 10.1186/s12872-024-04000-z

Figure Lengend Snippet: Fig. 6 XAV939 suppressed glycolysis and NLRP3 inflammasome activation in vivo. A Western blot showing the expression of β-catenin,HK2,PKM2 in vivo. Quantitative Western blot results were normalized to those of β-actin. B The glucose content in lung tissue from the four groups of rats. C-E The contents of PFK, LDH and LA in vivo. F Representative Western blot showing the expression of NLRP3 inflammasome markers in lung samples from the different groups. The blots were cropped and hybridized with the corresponding antibodies. β-Actin was used as the internal reference. G-J The levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β and IL-18 in the lung tissue supernatants of the rats. The following comparisons were made: Sham vs. Sham + XAV939, Sham vs. MCT, and MCT vs. MCT + XAV939. The data are presented as the means ± SDs; ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3/group

Article Snippet: The membranes were probed with antibodies against β-catenin (1:1000, Proteintech, Wuhan, China), HK2, PKM2 (1:4000, Proteintech, Wuhan, China), ASC, procaspase-1, caspase-1, NLRP3 (1:1000; all Cell Signaling Technology, USA), β-actin (1:2500, Proteintech, Wuhan, China).

Techniques: Activation Assay, In Vivo, Western Blot, Expressing

Journal: bioRxiv

Article Title: Functional regulation of 4D metabolic network between multienzyme glucosome condensates and mitochondria

doi: 10.1101/2022.05.16.491844

Figure Lengend Snippet: (a-c) A representative Hs578T cell showing 3D distribution of glucosomes (PFKL-mEGFP, green) relative to mitochondria (MitoTracker-Red, red) under LLSM imaging. Scale bars in (a) and (c), 5 µm and 1 µm, respectively. (d) Percentages of cells showing decreased, increased, and unchanged amounts of glucosomes at single cell levels in the absence (green) and presence (red) of oligomycin A (10 µM) ( N cells = 145). Error bars indicate standard errors. (e-f) Representative cropped regions of interest in the cytoplasm of a live Hs578T cell showing relative spatial distribution of glucosomes (green) and mitochondria (red) in the absence (e) and presence (f) of oligomycin. Scale bars, 1 µm. (g) Percentages of cells showing decreased, increased, and unchanged amounts of glucosomes at single cell levels in the absence (green) and presence (red) of a PDC inhibitor ((6,8-Bis[(phenylmethyl)thio]octanoic acid (CPI 613), N cells = 74). (h-j) A representative Hs578T cell showing spatial distributions of glucosomes (green) and mitochondria (red) after treatment of shRNA HKII . (i) and (j) show the side and the angled views of (h). Scale bars in (h) and (j), 5 µm and 1 µm, respectively. (k) Percentages of cells showing small-volume glucosomes with (red) and without (green) shRNA HKII treatment. Error bars show standard errors from at least 3 independent experiments.

Article Snippet: Lentiviral pGFP-shHKII vector encoding human shRNA HKII (Cat# TL312415) and non-silencing control pGFP-C-shLenti vector (Cat#: TR30023) were purchased from OriGene Technologies Inc (Rockville, MD, USA).

Techniques: Imaging, shRNA

Journal: bioRxiv

Article Title: Functional regulation of 4D metabolic network between multienzyme glucosome condensates and mitochondria

doi: 10.1101/2022.05.16.491844

Figure Lengend Snippet: (a) A histogram of glucosome populations (%) vs. the nearest edge-to-edge distance between glucosomes and mitochondria ( N glucosomes = 2147). A black solid line shows a double exponential fit for the histogram with a function ( f(d) = f(0) + A 1 e -δ1·d + A 2 e -δ2·d ) where mean distances for two subpopulations are expressed as 1/ δ1 and 1/ δ2 . A white arrow as shown in inset represents an example of the nearest edge-to-edge distance between a mitochondrion and a glucosome. (b) Histograms of glucosome populations (%) as a function of the nearest distance between glucosomes and mitochondria in mitochondria-dense (green) and mitochondria-sparse (red) regions ( N glucosomes = 899). (c-d) Histograms of populations of small-volume (< 90 voxels) and large-volume (≥ 90 voxels) glucosomes vs. the nearest distance between glucosomes and mitochondria only from mitochondria-dense regions in the presence (red) and absence (green) of oligomycin A treatment ( N glucosomes = 964). Error bars show standard errors. (e) Volume percentages (%) occupied by small-volume glucosomes at single cell levels with (red) ( N cells = 7) and without (green) shRNA HKII treatment ( N cells = 8). (f) A histogram of glucosome populations (%) vs. the nearest distance between glucosomes and mitochondria after treatment of shRNA HKII ( N glucosomes = 656). (g-h) Normalized apparent concentrations of PFKL and PKM2 as a function of glucosome volume. Note that the normalized apparent concentrations of PFKL and PKM2 are scaled based on the ratio of their partition coefficients . Statistical analyses were performed using a one-way analysis of variance (ANOVA). Statistical significance is defined as p < 0.05 with a 95 % confidence interval while ‘ns’ refers to not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars indicate standard errors from at least 3 independent experiments.

Article Snippet: Lentiviral pGFP-shHKII vector encoding human shRNA HKII (Cat# TL312415) and non-silencing control pGFP-C-shLenti vector (Cat#: TR30023) were purchased from OriGene Technologies Inc (Rockville, MD, USA).

Techniques: shRNA

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: Differential mRNA expression profiles of key glycolytic enzymes in silicotic rat lungs. Hk2 /HK2: hexokinase2, Gpi /GPI: glucose-6-phosphate isomerase, Pfkm : phosphofructokinase muscle, Pfkl : phosphofructokinase liver type, Pfkp : phosphofructokinase platelet, Aldoa /ALDOA: fructose-bisphosphate aldolase A, Gapdh /GAPDH: glyceraldehyde-3-phosphate dehydrogenase, Pgk1 /PGK1: phosphoglycerate kinase1, Pgam1 /PGAM1: phosphoglycerate mutase1, Eno1 /ENO1: enolase1, Pkm /PKM: pyruvate kinase M, Ldha /LDHA: lactate dehydrogenase A, PFK: phosphofructokinase. * Compared with control group, p < 0.05. C: control 24 w; S: silicosis 24 w.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: High expression of glycolysis in rats exposed to silica. ( A ) Positive expression of LDHA in silicotic rats observed by IHC staining, bars = 200 μm or 50 μm. ( B ) The co-expression of CD68 and LDHA in silicotic rats was measured by IF staining, bar = 50 μm. ( C ) Protein and mRNA expression levels of HK2, PKM2, and LDHA in silicotic rats measured by Western blotting and qRT-PCR. Data are presented as the mean ± SD. n = 4 per group. C: control 24 w; S: silicosis 24 w.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: Enhanced glycolysis is the crucial metabolic characteristic of silicotic mice. ( A ) Expression of LDHA in silicotic mice measured by IHC staining, bars = 200 μm or 50 μm. ( B ) Protein and mRNA expression levels of HK2, PKM2, and LDHA in mouse lungs measured by Western blotting and qRT-PCR. Data are presented as the mean ± SD. n = 5 or 4 per group. C: control 8 w; S: silicosis 8 w.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: Silica treatment increases the level of glycolysis in macrophages. ( A ) Changes in LDHA expression in NR8383 cells treated with 50, 100, 200, and 250 µg/mL silica measured by IF staining, bar = 50 μm. ( B ) Levels of HK2, PKM2, LDHA, iNOS, TNF-α, Arg-1, IL-10, and MCP1 in NR8383 cells treated with 50, 100, 200, and 250 µg/mL silica measured by Western blotting. * Compared with control group, p < 0.05. Data are presented as the mean ± SD. n = 4 per group. ( C ) The lactate content in the culture medium was detected using a lactate assay kit. *Compared with control group, p < 0.05.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Expressing, Staining, Western Blot, Control, Lactate Assay

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: Ac-SDKP attenuates the enhancement of glycolysis in macrophages treated with silica. ( A ) The co-expression of MCP1 and LDHA in NR8383 cells was measured by IF staining, bar= 50 μm; ( B ) The co-expression of Arg-1 and LDHA in NR8383 cells was measured by IF staining, bar= 50 μm. ( C ) Protein expression of HK2, PKM2, LDHA, iNOS, TNF-α, Arg-1, IL-10, and MCP1 in NR8383 cells treated with or without SiO 2 , Ac-ADKP, and Ac-SDKP. Data are presented as the mean ± SD. n = 3 per group. ( D ) The lactate content in the culture medium was detected using a lactate assay kit. C: control; S: SiO 2 ; AA: Ac-ADKP: SiO 2 and Ac-ADKP; Ac: Ac-SDKP: SiO 2 and Ac-SDKP.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Expressing, Staining, Lactate Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

doi: 10.3390/ijms221810063

Figure Lengend Snippet: Ac-SDKP attenuates high glycolytic activity and macrophage activation in rats exposed to silica. ( A ) The expression of LDHA in rats exposed to silica was measured by IF staining, bar = 50 μm. ( B ) The lactate content in rat lung tissue was detected using a lactate assay kit. ( C ) Levels of HK2, PKM2, LDHA, iNOS, TNF-α, Arg-1, IL-10, and MCP1 in silicotic rats were measured by Western blotting. Data are presented as the mean ± SD. n = 3 per group. C: control 24 w; S: silicosis 24 w; Ac: Ac-SDKP treatment 24 w.

Article Snippet: Western blotting was performed using published protocols [ , , ] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).

Techniques: Activity Assay, Activation Assay, Expressing, Staining, Lactate Assay, Western Blot, Control

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 2. Expression levels of glycolysis regulation proteins in SW480 and SW620 cells with different metabolic types. (A and B) Representative western blotting images of HIF-1α, HK1, HK2 and GLUT1 protein expressions in (A) SW480 and (B) SW620 cells cultured in different media. (C-F) Protein expression levels from (A) and (B) were quantified using Image Lab analysis software; the relative expression levels of HIF-1α, HK1, HK2 and GLUT1 were calculated as a ratio of OXPHOS; β-actin was used as the loading control. (G) Western blot analysis of HK1 and HK2 protein expression levels in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. (G) Representative western blots indicating the expression of HK1 and HK2 in SW480 and SW620 cells at different treatment times. (H and I) Protein expression levels from (G) were quantified using Image Lab analysis software, and relative expression levels of (H) HK1 and (I) HK2 were calculated as a ratio of 0 h; β-actin was used as a loading control. Data are presented as the mean ± standard deviation; n=3; **P<0.01 and ***P<0.001 vs. SW480. GLUT1, glucose transporter type 1; HIF-1α, hypoxia-inducible factor; HK, hexokinase; NOR, normal; GLY, glycolysis; OXPHOS, oxidative phosphorylation.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Cell Culture, Software, Control, Standard Deviation, Phospho-proteomics

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 6. RT-qPCR analysis of SW480 and SW620 cells in different culture microenvironments. (A-C) RT-qPCR analysis of the relative mRNA expression levels of HK1, HK2, HIF-1α, CDH1, FN1, VIM, CD133, SNAI1, SNAI2, ZEB1, CDH2 and TWIST1 in SW480 and SW620 cells cultured in different media conditions; mRNA expressions were normalized to β-actin. (D) SW620/SW480 mRNA expression ratios in different types of metabolism. Data are presented as the mean ± standard deviation; n=3; *P<0.05, **P<0.01 and ***P<0.001 vs. SW480. CDH1, Epithelial cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; Oligo A, Oligomycin A; OXPHOS, oxidative phosphorylation; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox; NOR, normal; GLY, glycolysis; OXPH, oxidative phosphorylation.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Standard Deviation, Phospho-proteomics, Binding Assay

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 7. mRNA levels in SW480 and SW620 cells treated with Oligo A over time. Reverse transcription-quantitative polymerase chain reaction analysis of mRNA expression levels of HK1, HK2, HIF-1α, CDH1, CDH2, ZEB1, SNAI1, SNAI2, TWIST1, CD133, VIM and FN in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. mRNA expression levels were normalized to β-actin. Data are presented as the mean ± standard deviation; n=3; **P<0.01; ***P<0.001 vs. SW480. CDH1, epithelial cadherin; CDH2, Neural-cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Binding Assay

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Forecasting the target gene by miR-125a-5p, the expression and biological role of HK2 in LSCC: (A) The research involved examining three sets of data, which involved predicting target genes controlled by miR-125a-5p, (B) creating the network of PPI and finding the important genes, (C) pinpointing the exact location where HK2 and miR-125a-5p bind, and (D, E) comparing levels of HK2 in laryngeal cancer tissues to nearby healthy tissues using immunohistochemical analysis. (F, G) Western blotting was performed to assess the HK2 expression levels in laryngeal cancer tissues compared to nearby non-cancerous tissues. (H) RT-PCR was used to assess HK2 expression levels in laryngeal cancer tissues in comparison to nearby non-cancerous tissues. (I, J) Furthermore, RT-PCR was performed to evaluate the effect on HK2 expression levels in AMN-HC-8 cells when either increased or decreased. (K) The ability of proliferation in laryngeal cancer cells was evaluated by using the CCK-8 assay after altering the expression levels of HK2. (L) The clonogenic assay was employed to investigate alterations in clonability observed in laryngeal cancer cells following modulation of HK2. (M) The dose survival curve demonstrated variations in surviving fraction resulting from up-regulation or down-regulation of HK2. (N) The comprehensive analysis was conducted using single hit multi-target model to investigate the impact of modulating HK2 expression levels on radiotherapy sensitivity. (O) Moreover, the luciferase test was used to verify the specific interaction between miR-125a-5p and HK2. (P, Q) Flow cytometry was used to confirm the impact of modified HK2 expression on apoptosis control. ( * P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, CCK-8 Assay, Clonogenic Assay, Luciferase, Flow Cytometry, Modification, Control

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Single hit multi-target model was used to analyze the effect of HK2 on the radiosensitivity of laryngeal cancer.

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques:

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: MiR-125a-5p regulates LSCC biological function and radiosensitivity by targeting HK2: (A) The regulation of LSCC cell proliferation is specifically mediated by miR-125a-5p through HK2 targeting. (B) This targeting of HK2 by miR-125a-5p impacts the clonogenic potential of LSCC cells. (C) Additionally, miR-125a-5p plays a role in determining the survival of laryngeal cancer cells under varying doses of irradiation by targeting HK2. (D) The single hit multi-target model approach was employed to assess alterations in the radiosensitivity of laryngeal cancer cells. (E, F) MiR-125a-5p modulates apoptosis in laryngeal cancer cells by interacting with HK2. (G–I) MiR-125a-5p regulates the levels of H2AX and rH2AX by targeting HK2. ( * P <0.05, ** P <0.01, and *** P <0.001).

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques: Irradiation

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Single hit multi-target model was used to analyze the effect of miR-125a-5p targeting HK2.

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques: