Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 4. HA344-induced cell death in CMM is initiated by PKM covalent targeting. A, Chemical structures of VG41 and VG43. B, Cell viability measured by trypan blue exclusion of A375S, A375R#1, A375R#2, SKMEL28, and SKMEL28R CMM treated with HA344, VG41, or VG43 at 2 mmol/L for 48 hours. C, Number of peptides according to peptide intensity of A375S cells click chemistry treated 1 hour with VG41 at 2 mmol/L. D, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of A375S and A375R#1 cell click chemistry. E, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of SKMEL28 and SKMEL28R cell click chemistry. F, Table of mass spectrometry analysis of recombinant PKM2 treated or not with HA344 (2 mmol/L, 1 hour). G, PKM2 and GAPDH Western blot analysis of A375S cell CETSA and their quantification. H, PKM2 and GAPDH Western blot analysis of A375R#1 cell CETSA and their quantification. , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Western Blot, Mass Spectrometry, Recombinant

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 5. HA344 inhibits glycolytic and nonglycolytic functions of PKM2 and IMPDH activity in CMM. A, PK enzymatic activity under HA344 (at 0.5, 1, and 2 mmol/L) and Shikonin (at 2 mmol/L) treatment. B, PK enzymatic activity of HEK293T cells of EV, PKM2, or PKM2 C424 L after GFP trap treated or not with HA344. C, PKM2 monomer and dimer Western blot analysis of A375S and A375R#1 cells treated 30 minutes, 3 hours, or 6 hours with HA344 at 2 mmol/L. D, Immunofluorescence PKM2 staining of A375S treated 30 minutes, 1 hour, and 6 hours with HA344 at 2 mmol/L, measured by confocal imaging. E, Cell-cycle analysis of A375R#1 treated 6 hours with HA344 at 2 mmol/L (top) and cyclin D1 Western blot analysis of A375S and A375R#1 (bottom). F, Glycolysis relative to control metabolite levels of A375S (top) and A375R#1 (bottom) treated 3 or 6 hours with HA344 at 2 mmol/L and 3 hours with Shikonin at 2 mmol/L. G, Unlabeled (Mþ0) and 13C-labeled glucose 6-P (Mþ6; left) and pyruvate (Mþ3; right) of A375S cells treated or not with HA344 at 2 mmol/L 3 hours and 6 hours and Shikonin 2 mmol/L. H, Unlabeled (Mþ0) and 13C-labeled glucose 6-P (Mþ6; left) and pyruvate (Mþ3; right) of A375R#1 cells treated or not with HA344 at 2 mmol/L 3 hours and 6 hours and Shikonin 2 mmol/L. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Activity Assay, Western Blot, Staining, Imaging, Cell Cycle Assay, Control, Labeling

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 6. HA344 reduces tumor growth in mice xenografted with CMM. A, A375S tumor growth in control and HA344-treated mice at 5 mg/kg/day. Tumor size is presented as the mean SEM, with n ¼ 6 mice in each group. Statistical significance was assessed by a two-way ANOVA with a Sidak multiple comparisons test. B, A375R#2 tumor growth in control and HA344-treated mice at 5 mg/kg/day). Tumor size is presented as the mean SEM with n ¼ 6 mice in each group. Statistical significance was assessed by a two-way ANOVA with a Sidak multiple comparisons test. C, Frozen sections of A375S tumors from mice treated with vehicle or HA344 were fixed and stained using PKM2 antibody (Alexa 594) and DAPI (blue). The experiment was repeated on sections from all mice per condition. Quantifications of PKM2 fluorescence intensity are presented in a.u. and represent the mean/field SEM of at least four independent fields. Statistical significance was assessed by an unpaired t test. D, Frozen sections of A375R#2 tumors from mice treated with vehicle or HA344 were fixed and stained using PKM2 antibody (Alexa 594) and DAPI (blue). The experiment was repeated on sections from all mice per condition. Quantifications of PKM2 fluorescence intensity are presented in a.u. and represent the mean/field SEM of at least four independent fields. Statistical significance was assessed by an unpaired t test. E, Quantification of PKM2 Western blot analysis of A375S xenografts in CT NaCl and HA344-treated tumors. F, Quantification of PKM2 Western blot analysis of A375R#2 xenografts in CT NaCl and HA344-treated tumors. G, Three mg of A375S tumor proteins from control (n ¼ 6) and HA344 (n ¼ 5)-treated groups were used to measure PKM activity (n ¼ 3 measures for each tumor). PKM activity is presented in RFU and as the mean SEM of tumors. H, Three mg of A375R#2 tumor proteins from control (n ¼ 6) and HA344 (n ¼ 5)-treated groups were used to measure PKM activity (n ¼ 3 measures for each tumor). PKM activity is presented in RFU and as the mean SEM of tumors. , P < 0.05; , P < 0.001; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Control, Staining, Western Blot, Activity Assay

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 7. HA344 induces cell death and targets PKM in patients with CMM. A, The SKCM TCGA dataset from Oncolnc was analyzed for PKM mRNA expression and OS. Patients were stratified according toPKM expression and BRAFV600E mutation. B, TheGSE3189 TCGA dataset from the GEO database was analyzed for PKM mRNA expression according to melanocyte transformation status. Statistical significance was determined using an unpaired t test. C, DAPI staining of CMM patients’short-term cultures treated 48 hours with vemurafenib (Vemu). D, DAPI staining of CMM patients’ short-term cultures treated 48 hours with HA344. E, Cell viability measured by trypan blue exclusion of patient#1, patient#6S, and patient#6R treated with HA344, VG43, and VG41. F, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of patient#1 click chemistry. G, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of patient#6S and patient# 6R click chemistry. H, Confocal images of PKM2 staining of patient#1 cells treated 30 minutes, 1 hour, and 6 hours with HA344 at 2 mmol/L. , P < 0.05; , P < 0.01; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Expressing, Mutagenesis, Transformation Assay, Staining, Western Blot

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Representative immunofluorescence images and quantification of glycolytic enzymes PKM2 and LDHA in human PLL and OPLL tissues. Scale bar = 100 µm, n = 3. B Western blot analysis of GLUT1, PKM2, and LDHA protein levels in PLL and OPLL cells. n = 3. C , D . Glucose uptake and lactate production in PLL and OPLL cells. n = 3. E ECAR measured by Seahorse XF Analyzer to assess the glycolytic capacity of PLL and OPLL cells. n = 3. F Representative images showing TMRE staining (orange-red) in PLL and OPLL cells. The bar graph depicts the quantification of relative TMRE fluorescence intensity. Scale bar = 30 µm, n = 3. G Western blot analysis of PDK1, Cyto c, and ATP5A in OPLL and PLL cells. H Intracellular ROS levels measured by DCFH-DA fluorescence. Scale bar = 50 µm. I OCR measured by Seahorse XF Analyzer to assess mitochondrial respiration. n = 3. J Western blot analysis of RUNX2, OSX, and ALP during a 15day time course of osteogenic differentiation of ligament cells. K Western blot analysis of GLUT1, HK2, and LDHA during a 15day time course of osteogenic differentiation of ligament cells. L Western blot analysis of PDK1, Cyto c, and ATP5A during a 15day time course of osteogenic differentiation of ligament cells. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Immunofluorescence, Western Blot, Staining, Fluorescence, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Venn diagram illustrating the overlap of hub genes identified from the PPI network using five different centrality algorithms: MCC, DMNC, EPC, MNC, and Degree. B Key genes identified by the DMNC method within the PPI network. C Correlation analysis between the glycolysis flux score and ADAM12 expression in OPLL and PLL tissues. D Distribution of ADAM12 across the major cell types in posterior longitudinal ligament tissue. E Scatter plot showing the positive correlation between ADAM12 expression and the glucose metabolism pathway activity score across individual ligament cells. F Representative immunofluorescence images and quantification of ADAM12 expression in human OPLL and PLL tissues. Scale bar = 50 μm, n = 3. G Representative immunofluorescence images showing co-localization of ADAM12 (green) and PKM2 (red) in OPLL tissue. Scale bar = 30 μm. H , I Quantitative PCR analysis of ADAM12L and ADAM12S mRNA levels in PLL and OPLL cells. n = 3. J Quantitative PCR analysis of the expression levels of the ADAM12L and ADAM12S isoforms in OPLL cells. n = 3. K Western blot analysis of ADAM12 protein levels in PLL and OPLL cells. n = 3. L ELISA analysis of ADAM12 protein levels in PLL and OPLL ligament cells supernatant. n = 5. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Expressing, Activity Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Heatmap showing the relative abundance of central carbon metabolites in control (NC), osteogenically differentiated (NC-OD), and ADAM12-overexpressing osteogenically differentiated (OE-OD) ligament cells. n = 3. B Box plots quantifying the levels of key glycolytic intermediates (fructose-1,6-bisphosphate, 3-phosphoglycerate, pyruvate, and lactate) from the metabolomics data. n = 3. C , D Western blot analysis of glycolytic markers (GLUT1, HK2, PKM2, LDHA) and mitochondrial markers (PGC1α, mtTFA, ATP5A, cytochrome c) in ligament cells after ADAM12 knockdown and overexpression. n = 3. E , F Glucose uptake capacity in ligament cells after ADAM12 knockdown and overexpression. n = 3. G , H Lactate production in ligament cells after ADAM12 knockdown and overexpression. n = 3. I , J Intracellular pyruvate content in ligament cells after ADAM12 knockdown and overexpression. n = 3. K , L ECAR measured by Seahorse XF Analyzer in ligament cells after ADAM12 knockdown and overexpression. n = 3. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Control, Western Blot, Knockdown, Over Expression, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Representative images comparing whole kidney size between Glis3-KO2 kidneys treated with vehicle or compound 3K are shown. b , Violin plot depicting the KW/BW ratio (%) for WT and Glis3 -KO2 mice treated with vehicle or compound 3K. n ≥ 10; **** P < 0.0001. c , Representative hematoxylin and eosin-scanned images of kidney sections from Glis3 -KO2 kidneys treated with vehicle or compound 3K. Bars indicate 1 mm. d , Comparison of cystic index between Glis3 -KO2 kidneys treated with vehicle or compound 3K. Cystic index represents the percentage of renal tissue occupied by cysts. Data are presented as mean ± s.e.m., n = 6 . ** P < 0.01. e , Comparison of renal cyst size (mm 2 ) between kidneys from Glis3 -KO2 mice treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n = 6; * P < 0.05. f , Comparison of the number of cysts per kidney section between Glis3 -KO2 kidneys treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n = 8; ** P < 0.01. g , Comparison of serum creatinine levels (mg/dl) between WT and Glis3 -KO2 mice treated with vehicle or compound 3K. h , RT–qPCR analysis of Pkm2 , c-Myc , Hk2 , Havcr1 and Lcn2 between WT and Glis3 -KO2 mice treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n ≥ 5; **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. i , Schematic illustration depicting the relationship between loss of GLIS3 function, regulation of PKM2 and cystogenesis. GLIS3 deficiency enhances Pkm gene expression in kidneys with a preferential increase in the Pkm2 isoform. Increased PKM2 phosphorylation at S37 and Y105 promotes dimer formation. PKM2-S37 phosphorylation is facilitated by increased pERK1/2 levels. Together, these events promote glycolysis, cell proliferation and cystogenesis in GLIS3-deficient kidneys. Figure 6i was created using BioRender.com.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Comparison, Quantitative RT-PCR, Gene Expression, Phospho-proteomics

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , b , Analysis of RNA-seq transcripts per million (TPM) values for PKM2 transcripts ( a ) and PKM2:PKM1 transcript ratio ( b ) in PND7, 14 and 28 WT and Glis3 -KO2 kidneys. c , Sashimi analysis plot showing the alternatively spliced isoforms of PKM2 and PKM1. Exons (darker color) and splice junctions (lighter color) from WT are in blue and those from Glis3 -KO2 are in purple. Splice junction read counts are numbered above their respective ribbons. STAR genomic alignment counts scaled to 300 M mappable reads per sample group labels the y axis, exon and splice junction coordinates are on the x axis and mRNA isoforms are shown on the bottom (exons in black and introns as lines). d , Analysis of the PKM2-specific splice junction read counts, plotted for visualization between PND7, 14 and 28 WT and Glis3 -KO2 kidneys. e , f , Exogenous expression of GLIS3 in WT and Glis3 -KO2 RECs decreased Pkm2 mRNA expression ( e ) but did not change Pkm1 mRNA expression ( f ). RECs were infected with Glis3 lentivirus for 36 h, and gene expression was analyzed by RT–qPCR. Data are presented as mean ± s.e.m., n ≥ 5 ; **** P < 0.0001, *** P < 0.001; ** P < 0.01; * P < 0.05. n.s., nonsignificant. g , RT–qPCR for Pkm2 from isolated collecting duct (CD) and proximal tubule (PT) cells. Data are presented as mean ± s.e.m., n ≥ 3; * P < 0.05.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: RNA Sequencing, Expressing, Infection, Gene Expression, Quantitative RT-PCR, Isolation

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Immunoblot analysis of total PKM2 (GA−) and glutaraldehyde (GA+) crosslinked PKM2 expression. The relative level of PKM2 dimers were quantified by densitometric analysis. b , Immunoblot analysis of PKM2(pY105) and total PKM2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 3 ; ** P < 0.01; * P < 0.05. c , Immunoblot analysis of PKM2(pS37) and total PKM2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 3 ; ** P < 0.01; * P < 0.05. d , Representative images of PND28 WT and Glis3 -KO2 kidney sections immunostained for PKM2(pS37) showing enhanced nuclear staining in renal cysts. The inlets denote the location of zoomed-in images. DBA marks collecting ducts and LTL marks proximal tubules. e , Immunoblot analysis of pERK1/2 and total ERK1/2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 4; ** P < 0.01.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Western Blot, Expressing, Staining

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Immunoblot analysis of PKM2 protein levels in WT and Glis3 -KO2 RECS 3 days after siRNA-mediated PKM2-KD. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 5; *** P < 0.001; ** P < 0.01. b , Analysis of lactate production in media from primary WT and Glis3 -KO2 RECs with or without PKM2-KD. c , Glycolytic rate was measured in primary WT and Glis3 -KO2 REC mice with or without PKM2-KD using a Seahorse analyzer after sequential injections of rotenone/antimycin A and 2-DG. d , e , Basal ( d ) and compensatory ( e ) glycolysis were calculated and plotted ( n = 3). f , Representative images of WT and Glis3 -KO2 REC spheroids with and without PKM2-KD at 5 and 9 days. Bars indicate 50 μm. g , Violin plot showing the size (µm) distribution of the spheroids generated at day 5 from WT and Glis3 -KO2 RECs with or without PKM2-KD ( n ≥ 4). Each data point represents an individual spheroid measurement. * P < 0.05, ** P < 0.01, *** P < 0.001. h , Spheroid images at day 5 were taken using the EVOS M7000, and spheroid diameter and number were analyzed using ImageJ and plotted according to size distribution—either 30–50, 50–100 or >100 μm. Total indicates the number of spheroids analyzed in each group ( n = 39–164). i , Violin plot showing the size (µm) distribution of the spheroids generated at day 9 from WT and Glis3 -KO2 REC mice with or without PKM2-KD ( n ≥ 4). Each data point represents an individual spheroid measurement. * P < 0.05, ** P < 0.01, *** P < 0.001. j , Day 10 spheroid size distribution—either 30–50, 50–100 or >100 μm. Total indicates the number of spheroids analyzed in each group ( n = 60–191). k , Representative image of the size of WT and Glis3 -KO2 REC spheroids 5 days following treatment with vehicle (0.1% DMSO) or compound 3K (1 μM). Bars indicate 50 μm. l , Violin plot showing the size (µm) distribution of the spheroids generated from the RECs of WT and Glis3 -KO2 kidneys ( n ≥ 4). Each data point represents an individual spheroid measurement. **** P < 0.0001. m , Size distribution—30–50, 50–100 or >100 μm of WT and Glis3 -KO2 REC spheroids with or without PKM2 inhibition. Total indicates the number of spheroids analyzed in each group (n = 39–164).

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Western Blot, Expressing, Generated, Inhibition

Journal: Oncology reports

Article Title: MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

doi: 10.3892/or.2015.4175

Figure Lengend Snippet: Figure 4. PKM2 is a direct target of miR-122. (A) Target prediction from TargetScan.org, indicates that the position 520-527 of PKM2 3'-UTR contains putative binding sites for miR-122. (B) Effects of miR-122 suppression or overexpression on endogenous PKM2 levels. Forty-eight hours after transfection as shown, the Hep3B cells were harvested and analyzed by western blotting. (C) Luciferase assays of cells cotransfected with miR-NC (negative control) or pre-miR-122, firefly luciferase reporter plasmid and pRL-TK containing the mutant 3'-UTR of PKM2 or wild-type. (D) Luciferase assays of cells transfected with anti‑miR-122 or anti-miR‑NC, pRL-TK and the firefly luciferase reporter comprising the wild-type or mutant 3'-UTR of PKM2 siRNA. We cotransfected pRL-TK which expresses Renilla luciferase as an internal control, and each sample's firefly luciferase activity was normalized to the Renilla luciferase activity. The normalized luciferase activity of anti-miR-NC transfectants or miR-NC was set as relative luciferase activity 1, therefore no error bar was shown for miR-NC and anti-miR-NC transfectants. *P<0.05, compared with the anti-miR-NC transfectants or miR-NC. 3'-UTR, 3'-untranslated region.

Article Snippet: The overexpression vectors containing wild-type PKM2 (Myc-DDK-tagged) were purchased from OriGene. siRNA PKM2 was obtained from Santa Cruz Biotechnology (sc-62820; Santa Cruz, CA, USA).

Techniques: Binding Assay, Over Expression, Transfection, Western Blot, Luciferase, Negative Control, Plasmid Preparation, Mutagenesis, Control, Activity Assay

Journal: Oncology reports

Article Title: MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

doi: 10.3892/or.2015.4175

Figure Lengend Snippet: Figure 3. miR-122 levels are negatively correlated with PKM2 expression in the HCC tissues. (A) Representative images of immunohistochemical staining for PKM2. Magnification, x400. Scale bar, 50 µm. (B) HCC tumors which had a lower miR-122 level showed higher PKM2 expression. Low-miR-122 (n=48) and high-miR-122-expressing tumors (n=12) were distinguished as described in Fig. 2. Expression of PKM2 was quantified in the immunohistochemical staining using an IOD by Image-Pro Plus 6.0 software. For B the Mann-Whitney test is shown. The central horizontal line suggests the mean value and the error bar indicates SEM (*P<0.05). HCC, hepatocellular carcinoma.

Article Snippet: The overexpression vectors containing wild-type PKM2 (Myc-DDK-tagged) were purchased from OriGene. siRNA PKM2 was obtained from Santa Cruz Biotechnology (sc-62820; Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Software, MANN-WHITNEY

Journal: Oncology reports

Article Title: MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

doi: 10.3892/or.2015.4175

Figure Lengend Snippet: Figure 5. miR-122 inhibits the apoptosis of Hep3B cells in a PKM2-dependent manner. (A) Apoptosis analysis of Hep3B cells transfected with anti-miR-122 and its scramble control. Apoptotic cells were assessed by flow cytometry. Anti-miR-122 reduced the percentage of apoptotic cells. *P<0.05 by one-way ANOVA; n=6 repeats with similar results. (B) Western blot analysis indicate the effect of siRNA PKM2 on PKM2 expression in Hep3B cells. (C) Apoptosis analysis of Hep3B cells transfected with anti-miR-122 and siRNA PKM2. Histogram shows that the apoptotic rate was obviously increased compared to the apoptotic rate in the anti‑miR-122 transfection group. *P<0.05 by one-way ANOVA; n=6 repeats with similar results. (D) siRNA PKM2 reduced the viability of anti-miR-122 in Hep3B cells. *P<0.05 by two-way ANOVA. Data are expressed as the mean ± SEM.

Article Snippet: The overexpression vectors containing wild-type PKM2 (Myc-DDK-tagged) were purchased from OriGene. siRNA PKM2 was obtained from Santa Cruz Biotechnology (sc-62820; Santa Cruz, CA, USA).

Techniques: Transfection, Control, Flow Cytometry, Western Blot, Expressing

Journal: Oncology reports

Article Title: MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

doi: 10.3892/or.2015.4175

Figure Lengend Snippet: Figure 6. miR-122 regulates the invasion and migration of Hep3B cells by targeting PKM2. (A) Hep3B cells transfected with the negative control and anti‑miR-122 were subjected to qRT-PCR for miR-122 (n=6; *P<0.05). (B and C) Wound healing assays revealed that knockdown of miR-122 increased the migration of Hep3B cells, while, siRNA PKM2 was found to reduced the inhibition effect on migration of anti-miR-122 in the Hep3B cells, *P<0.05 by t-test.

Article Snippet: The overexpression vectors containing wild-type PKM2 (Myc-DDK-tagged) were purchased from OriGene. siRNA PKM2 was obtained from Santa Cruz Biotechnology (sc-62820; Santa Cruz, CA, USA).

Techniques: Migration, Transfection, Negative Control, Quantitative RT-PCR, Knockdown, Inhibition

Journal: Oncology reports

Article Title: MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

doi: 10.3892/or.2015.4175

Figure Lengend Snippet: Figure 8. miR-122 suppresses tumor growth by targeting PKM2 in vivo. (A) Mice were treated with miR-122+PKM2, miR-122 or miR-control by multicenter intratumoral injection using an Hep3B subcutaneous tumor model (n=6, respectively). Tumor nodules were measured after different time periods (3, 6, 9, 12, 15, 18 and 21 day after implantation). miR-122-overexpressing Hep3B cells exhibited slower tumor growth ability in mice compared with the control group; however, re-expression of PKM2 partially restored tumor growth, when compared with the miR-122 group. *P<0.05 by two-way ANOVA. (B) Tumor nodules were subjected to immunohistochemical staining for TUNEL and Ki-67 assays. The results indicated that miR-122 overexpression induced apoptosis and inhibited proliferation in vivo. PKM2 not only partially abolished the inhibitory effect of miR-122 on HCC growth, yet also significantly reduced cell apoptosis (TUNEL assays) and increased the number of cells staining positive for Ki-67. Scale bar, 100 µm; n=6; data are expressed as the mean ± SEM; *P<0.05 by one-way ANOVA.

Article Snippet: The overexpression vectors containing wild-type PKM2 (Myc-DDK-tagged) were purchased from OriGene. siRNA PKM2 was obtained from Santa Cruz Biotechnology (sc-62820; Santa Cruz, CA, USA).

Techniques: In Vivo, Control, Injection, Expressing, Immunohistochemical staining, Staining, TUNEL Assay, Over Expression

Journal: Advanced Functional Materials

Article Title: Double Cross‐Linked Hydrogel for Intra‐articular Injection as Modality for Macrophages Metabolic Reprogramming and Therapy of Rheumatoid Arthritis

doi: 10.1002/adfm.202502880

Figure Lengend Snippet: Figure 4. Inhibition of glycolytic metabolism and M1 polarization of RAW264.7 macrophages. A) Schematic depiction of the inhibition of glycolysis pathway and subsequent blocking of M1 polarization. B) Glucose consumption and C) pyruvate production in RAW264.7 macrophages. The data are expressed as mean ± SD (n = 3) represented by error bars. D) Representative immunofluorescence micrographs of GLUT1 and HIF-1𝛼expression in RAW264.7 macrophages (scale bar, 20 μm). E) Western blots of HIF-1𝛼, GLUT1, and PKM2 expression in RAW264.7 macrophages. GAPDH, loading control. F) Relative expression of M1 macrophage markers detected in RAW264.7 macrophages. The data are expressed as mean ± SD (n = 3) represented by error bars. G) Flow cytometry dotplots of CD86 expression in RAW264.7 macrophages. Groups: I: ZA, II: Alg-siHPTs, III: siHPTs, IV: siHPTs@ZA hydrogel. The data are expressed as mean ± SD (n = 3), ****p < 0.0001.

Article Snippet: Next, membranes were blocked using BSA and incubated with specific primary antibodies: TNF alpha (1000×, ER1919-22, HUABIO), GLUT1, HIF-1α, PKM2 (1000×, 1F2-F4-H11, OriGene), PPARγ (5ab209350, Abcam) and GAPDH (1000×, AF3006, Beyotime) at 4 °C overnight.

Techniques: Inhibition, Blocking Assay, Western Blot, Expressing, Control, Flow Cytometry