Journal: Scientific Reports

Article Title: Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells

doi: 10.1038/s41598-023-36007-5

Figure Lengend Snippet: Inhibition of glycolysis reduced BM EPCs proliferation and inhibition of key enzymes of the PPP inhibited BM EPCs proliferation. ( A ) Schema for three key glycolytic enzymes, HK II, PFK and PKM, and their respective inhibitors, BP, 3PO and PKM2-IN-1. Schema for PPP enzyme, G6PD, and its respective inhibitor, 6AN. ( B ) Proliferation assay of BM EPCs treated with vehicle or various concentrations of BP under normoxia or hypoxia (n = 6). The number of EPC colonies in the presence of 100uM BP was significantly lower than that of the vehicle controls both under normoxia and under hypoxia (n = 6). ( C ) Proliferation assay of BM EPCs treated with vehicle or various concentration of 3PO under normoxia or hypoxia (n = 6). The number of BM EPCs colonies in the presence of 20uM 3PO was significantly lower than that of the vehicle controls both under normoxia and under hypoxia (n = 6). ( D ) Proliferation assay of BM EPCs treated with vehicle or various concentration of PKM2-IN-1 under normoxia or hypoxia (n = 6). The number of BM EPCs colonies in the presence of 10uM PKM2-IN-1 was significantly lower than that of the vehicle controls both under normoxia and hypoxia (n = 6). ( E ) Proliferation assay of BM EPCs treated with vehicle or various concentrations of 6AN under normoxia or hypoxia (n = 6). ( F ) The number BM EPCs colonies in the presence of 20uM 6AN was significantly lower than that of the vehicle controls both under normoxia and under hypoxia (n = 6). ( G ) Representative experiment showing ECAR of BM EPCs and comparison of glycolytic rate and capacity in BM EPCs treated with or without of 6AN under normoxia (n = 6). ( H ) Representative experiment showing OCR of BM EPCs and comparison of the OCR of basal and maximal respiration in the presence or absence of 6AN under normoxia. ( I ) Representative experiment showing ECAR of BM EPCs and comparison of glycolytic rate and capacity in the presence or absence of 6AN under hypoxia. ( J ) Representative experiment showing OCR of BM EPCs and comparison of basal and maximal respiration in the presence or absence of 6AN under hypoxia. Data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle control (with or without DMSO).

Article Snippet: The following inhibitors were used: BP (Sigma, 1113-59-3), 3PO (MCE, HY-19824), PKM2-IN-1 (MCE, HY-103617), 6-AN (Sigma, 329-89-5), UK5099 (MCE, HY-15475), AZD7545 (Selleck Chemicals, s7517), PCA (Sigma, 188174-64-3), SP (MCE, HY-12688A) and AA (Abcam, Ab141904).

Techniques: Inhibition, Proliferation Assay, Concentration Assay, Comparison, Control

Journal: Cell Death & Disease

Article Title: Inhibition of PFKP in renal tubular epithelial cell restrains TGF-β induced glycolysis and renal fibrosis

doi: 10.1038/s41419-023-06347-1

Figure Lengend Snippet: A , C Immunoblotting revealed the expression of glycolysis-related genes such as phospho-LDHA, HIF-1α, HEK2, and phospho-PKM2 in the renal tissues of mice in UUO group compared with sham group. The quantitative results of western blot are shown in the right panel, n = 6 (β-actin was used as the loading control). B Immunohistochemistry showed that phospho-LDHA, HIF-1α, and HEK2 in the renal tissues of mice in UUO group compared with sham group. D For immunohistochemistry staining detection for p-LDHA, HIF-1α, and HEK2 was quantified in the kidney sections in 3 fields per mice at ×100 magnification, n = 6. E Lactate concentrations in renal tubule cells from UUO group and sham group, n = 6. Data are shown as means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against FN1 (cat. #63779S), phospho-PKM2 (p-PKM2) (Tyr105) (cat. #3827S), PKM2 (cat. #4053), and LDHA (cat. #2012S), anti-mouse or rabbit IgG antibody were purchased from Cell Signaling Biotechnology (MA, USA).

Techniques: Western Blot, Expressing, Control, Immunohistochemistry, 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 , 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: The primary antibodies used in these studies included PKM2 (4053; Cell Signaling), pPKMS-S37 (11456; SAB), β-catenin (8480; Cell Signaling) and β-actin (MA5-15739; ThermoFisher).

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: The primary antibodies used in these studies included PKM2 (4053; Cell Signaling), pPKMS-S37 (11456; SAB), β-catenin (8480; Cell Signaling) and β-actin (MA5-15739; ThermoFisher).

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: The primary antibodies used in these studies included PKM2 (4053; Cell Signaling), pPKMS-S37 (11456; SAB), β-catenin (8480; Cell Signaling) and β-actin (MA5-15739; ThermoFisher).

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: The primary antibodies used in these studies included PKM2 (4053; Cell Signaling), pPKMS-S37 (11456; SAB), β-catenin (8480; Cell Signaling) and β-actin (MA5-15739; ThermoFisher).

Techniques: Western Blot, Expressing, Generated, Inhibition

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 4 PKM2 was a target of miR-1294 in osteosarcoma cells. (A) PKM2 expression in human mesenchymal stem cells (hMSCs) and five osteosarcoma cell lines (Saos-2, MG63, U2OS, HOS, and 143B) was assessed by real-time PCR. (B) The miR-1294 binding site predictions for PKM2 3ʹUTR by microRNA.org. (C) A dual luciferase reporter assay was performed to confirm the binding in 293T cells. (D and E) PKM2 expression in U2OS and HOS cells transfected with miR-1294 mimic by real-time PCR and Western blot assays. (F and G) PKM2 expression in 143B cells transfected with miR-1294 inhibitor by real-time PCR and Western blot assays. Data are shown as mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Reporter Assay, Transfection, Western Blot

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 5 Overexpression of PKM2 reversed the actions of miR-1294 upregulation on osteosarcoma cells. (A) Cell proliferation was evaluated by CCK-8 assay. (B) Cell apoptosis was examined by flow cytometry using Annexin Vand PI staining. (C) Cell migration was assessed by a wound-healing assay. (D) Cell invasiveness was determined by transwell assay. (E) Expression levels of c-Myc, cyclin D1, cleaved-caspase 3, MMP-2, and MMP-9 were texted by Western blotting. Data are shown as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Over Expression, CCK-8 Assay, Cytometry, Staining, Migration, Wound Healing Assay, Transwell Assay, Expressing, Western Blot

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 6 Overexpression of miR-1294 inhibited tumorigenicity of osteosarcoma cells. (A, B) Tumor volume in nude mice after subcutaneous injection of ME63 and U2O2 cells with or without overexpression of miR-1294. (C) The expression of miR-1294 in tumor tissues was determined by real-time PCR. (D, E) The expression of PKM2, c-Myc, cyclin D1, MMP-2, and MMP-9 in tumor tissues was assessed using immunohistochemistry and Western blot analysis. Data are shown as mean ± SD from 6 mice/ group. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Over Expression, Injection, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Western Blot

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 1. Rationale for genetic ablation of Pkm2 during HRas-driven nonmuscle-invasive bladder tumorigenesis. A, Changes in the expression of PKM2 protein during the sequential steps of nonmuscle-invasive bladder tumorigenesis induced by oncogenic HRas. Top, representative hematoxylin and eosin images of (from left to right) normal urothelium (NU) from a 6-month-old WT mouse and age-dependent, urothelial tumorigenesis in homozygous Upk2-HRas/ transgenic mice exhibiting SH (from a 1-month-old mouse), to NH (from a 2-month-old mouse) characterized by fibrovascular cores (arrows) within proliferative urothelia, and to NMBICs (from a 4-month-old and a 6-month-old mouse, respectively). Scale bars, 100 mm. Bottom, Western blotting (3 mice/genotype/time point corresponding to the top panel aligned by the dashed lines), showing relatively low PKM2 expression in normal urothelium, increased expression in simple hyperplasia, and marked and sustained increase in nodular hyperplasia and NMIBC (about 3 to 4-fold more compared with simple hyperplasia). Also note the lack of PKM1 overexpression in nodular hyperplasia and NMIBC. b-Actin (bottom) served as a loading control. The same membrane was stripped and reblotted with the different antibodies. M, months. B, Genotyping results of mouse tail DNAs from genetically engineered mouse lines. Three founder lines were used for intercrosses in order to produce compound mice that express oncogenic HRas and lack Pkm2 in a urothelium-specific manner, including (i) Upk2-HRas mice, (ii) Upk2-Cre mice, and (iii) “floxed” Pkm2flmice (16) in which exon 10 of the Pkm gene is flanked by the floxP sequences. The allelic status was verified by genomic PCR and additionally by qRT-PCR of the mouse tail DNAs to ascertain the homozygous status of the Upk2-HRas allele (bar diagram). The genotypes of one representative litter are shown, with four different genotypes chosen for the present work and abbreviated as: L1, WT; L2, Upk2-HRas//Pkm2fl/fl; L3, Upk2-Cre/Pkm2fl/fl; and L4, Upk2-HRas//Upk2-Cre/Pkm2fl/fl. C, qRT-PCR (top) and Western blotting (bottom) of PKM2 expression in urothelial cells establishing significantly elevated expression of PKM2 in 2-month-old L2 mice and markedly decreased expression of PKM2 in 2-month-old L4 mice. The small amounts of PKM2 in L4 mice were likely due to mesenchymal cells (which do not express Cre) of the tumor extracts (Fig. 2E).

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Expressing, Transgenic Assay, Western Blot, Over Expression, Control, Membrane, Quantitative RT-PCR

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 2. Phenotypic changes of urothelial tumorigenesis resulting from Pkm2 ablation. A, Urine dipstick test of hematuria (arrowhead denoting the sixth square from the top in each column for which the color changes from orange to dark blue indicates hematuria) showing lower frequency of hematuria in L4 mice than in L2 mice in all age groups (column/mouse; n ¼ 5/genotype/age group). B, Body and bladder weights at mouse sacrifice of L1, L2, and L4 (n ¼ 10/genotype/time point) showing reduced bladder weights in L4 mice at 4 and 6 months, as compared with age- and genotype-matched L2 mice. C, Representative gross anatomy images (n ¼ 2/genotype) showing much smaller bladders in L4 mice than in L2 mice (age-matched at 6 months). D, Representative images of hematoxylin and eosin–stained bladder sections (1 mouse per genotype; low magnification in the top panels and high magnification (five-fold of the top panels of the boxed areas in the lower panels) showing significantly reduced tumor masses of NMIBC from L4 mice as compared with those from the L2 mice (additional mice shown in Supplementary Fig. S1). Scale bar, 500 mm. E, Double immunofluorescent labeling showing (i) the increased expression of PKM2 and the number of Krt14-positive cells in L2 mice and the lack of expression of PKM2 (except the connective tissue, marked by circles with white-dashed lines) and the decreased number of Krt14-positive cells in L4 mice (both 6 months of age); (ii) moderately increased expression of PKM1 in PKM2-deleted tumor cells of L4 mice; and (iii) increased number of Ki67-positive cells in L2 mice (compared with L1 mice) and decreased number of ki67-positive cells in L4 mice (compared with L2). All the panels are of the same magnification. Scale bar, 100 mm. Semiquantification of Ki67-positive cells (percentage of DAPI-stained total urothelial cells counted) was presented in the diagram (lower right; , P < 0.05 between L4 and L2). M, months. , P < 0.05.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Staining, Labeling, Expressing

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 3. Key angiogenesis signals in PKM2- expressing and PKM2-lacking urothe- lial lesions. A, Western blotting show- ing low level of HIF1a in normal urothe- lium of L1 mice and the steadily increased expression of HIF1a in L2 mice particularly in mice exhibiting nodular hyperplasia (2 months) and NMIBC (6 months; see Fig. 1A for mor- phology and PKM2 expression). CD34 and N-cadherin overexpression was associatedprimarilywithnodularhyper- plasia (at 2 months of age) and NMIBC (6months). M, months. B, Western blots that showed markedly upregulated angiogenic signals (HIF1a, N-cadherin, VEGF, and CD34) in NMIBC of 6-month- old L2 mice, all of which were markedly downregulated in 6-month-old L4 mice lacking PKM2. C, Double immunofluo- rescent staining showing that while the CD34 and HIF1a were overexpressed and somewhat colocalized in the fibro- vascular cores of the NMIBC of L2 mice (6-month-old), they were significantly diminished in PKM2-deleted, 6-month- old L4 mice. All the panels are of the same magnifications. Scale bar, 100 mm. N-cad, N-cadherin.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Expressing, Western Blot, Over Expression, Staining

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 4. Expression and subcellular localization of STAT3 and phosphorylated STAT3 (Y705) in PKM2-expressing versus PKM2-lacking urothelial lesions. A, Western blotting assessing the levels of key downstream effectors of RAS pathway (both nonphosphorylated and phosphorylated forms), showing the marked overexpression as well as activation of ERK1/2, AKT, and STAT3 in NMIBC of the 6-month-old L2 mice and, in contrast, the marked and selective downregulation of activated ERK1/2 and STAT3. B, Subcellular fractionation and Western blotting showing that STAT3 and phosphorylated STAT3 (Y705), which did not differ significantly in the cytoplasmic fractions between PKM2-expressing (L2 mice) and PKM2-lacking (L4 mouse) urothelial lesions, significantly decreased in the nuclear fraction of PKM2-lacking urothelial lesions of the L4 mice. b-Actin and histone H3 served as loading controls for the cytoplasmic and nuclear proteins, respectively. C, Immunofluorescent staining showing the nuclear localization of activated STAT3 in NMIBC cells (arrows) of L2 mice, and the marked downregulation and absent nuclear location of activated STAT3 in PKM2-depleted urothelial lesions of L4 mice. Higher magnifications from the boxed areas of the top panels are shown in the bottom panels. Scale bars, 50 mm. D, Subcellular fractionation and Western blotting showing that, compared with the normal urothelium of the L1 mice (6-month-old), oncogenic HRas-expressing NMIBC cells of the age-matched L2 mice have elevated levels of PKM2 and phosphorylated PKM2 in the cytoplasm as well as in the nucleus. b-Actin and histone H3 served as loading controls for the cytoplasmic and nuclear proteins, respectively. H3, histone H3; p, phophorylated.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Expressing, Western Blot, Over Expression, Activation Assay, Fractionation, Staining

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 5. Effects of chemical inhibition of PKM2 on oncogenic HRas-induced NMBC. Ten L2 male mice (8weeks old) were randomized into two groups, one receiving DMSO only as a control and another receiving DMSO containing shikonin (2 mg/kg) intraperitoneally on days 1, 3, 5, 8, 10, and 12 before sacrifice on day 15 posttreatment. A, Gross anatomy showing bladders from control- and shikonin-treated mice (boxed images on the left) and tumor weights (bar diagram on the right; n ¼ 5/group). B, Hematoxylin and eosin–stained cross sections of bladders from control- and shikonin- treated mice (middle panels show higher magnification of the boxed areas of the left panels), and double IF staining of control- and shikonin- treated mice with anti-Hif1a and CD34 (right). Scale bars, 100 mm.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Inhibition, Control, Staining

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 6. Effects of time-controlled overexpression of Pkm1 and Pkm2 in NH in HRas/ mice. A, Two separate intercrosses were carried out in parallel, the first among Upk2- HRas, Upk2-rtTA, and Tre-Pkm1, and the second among Upk2-HRas, Upk2-rtTA, and Tre-Pkm2. Four lines with the following genotypes were chosen for phenotyping: L7, WT; L8, Upk2-HRas/; L9, Upk2-HRas and L10, Upk2-HRas//Upk2-rtTA/Tre-Pkm2. B, Western blotting showing overexpression of PKM1 and PKM2 in doxycycline-treated L9 and L10 mice, respectively, compared with doxycycline-treated L8 mice (after 4 weeks of treatment). C, Ultrasound images of age- (all 12 weeks of age), gender- (all male), and doxycycline treatment–matched (treated for 4 weeks) of the four genotypes described in A, showing smooth urothelium in a L7 WT mouse, nodular hyperplastic changes in L8 and L9 mice, and tumors occupying much of the bladder lumen in an L10 mouse. D, Three-dimonsional volume measurements of urothelia by ultrasound imaging (left; n ¼ 3/genotype) and bladder weights (right; n ¼ 5/genotype) illustrate more quantitatively the significantly greater urothelial volume and bladder weights in L10 mice than in L7, L8, and L9 mice. NS, not significant. E, Immunoprecipitation of STAT3 and PKM2 complex. Top, input proteins as assessed by Western blotting. Bottom three panels, anti-STAT3 pulled down PKM2 upon Western blotting; anti-PKM2 pulled down STAT3 upon Western blotting; and antiphosphorylated PKM2 (Y105) pulled down phosphorylated STAT3 (Y705). “þ” and “” denote the presence and absence (negative control), respectively, of the primary antibody during immunoprecipitation. WB, Western blotting; IP, immunoprecipitation. F, Paraffin-embedding sections stained by hematoxylin and eosin (H&E; left column) or IF (right two columns) showing (i) nodular hyperplasia in an L8 mouse and NIMBC in an L10 mouse (both 12-week-old males after 4 weeks of doxycycline treatment) and (ii) increased expression of angiogenic signals CD34 and Hif1a and proliferation/progenitor cell markers Ki67 and Krt14 in L10 mice, compared with L8 mice. Scale bars, 100 mm. p, phosphorylated.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Over Expression, Western Blot, Imaging, Immunoprecipitation, Negative Control, Staining, Expressing

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 7. Proximity ligation assay and chemical inhibition confirming the interaction between PKM2 and STAT3 and the importance of STAT3 activation in HRas-induced expression of angiogenicfactors. A,Top, positive controls provided by the commercialsupplier inwhich the cultured SK-OV3 human ovarian cancer cellswere treated with EGF to stimulate the binding between EGFR and HER2. Note that single immunofluorescent staining using anti-EGFR or anti-HER2 yielded little or no staining, whereas double immunofluorescent staining produced intense membranous/cytoplasmic staining, thus demonstrating the functionality of the system. Bottom, single or double immunofluorescent staining of normal mouse urothelium using a mouse anti-uroplakin (UP) Ib and/or a rabbit anti-UPIIIa, showing the absence of staining during single staining and the intense luminal urothelial staining during double staining, consistent with the known interaction between UPIb and UPIIIa. Scale bars, 10 mm. B, Double immunofluorescent staining in NMIBC from L10 mice (Fig. 6) using the antibodies indicated in each panel. M, mouse; p, phosphorylated; R, rabbit. Note the prominent complex formation between PKM2 and STAT3 as well as between p-PKM2 (Y105) and p-STAT3 (Y705), both primarily localized in the nuclei. Also note the lack of interaction between PKM2 and AKT and the relatively weak interaction between PKM2 and ERK1/2 primarily localized to the cytoplasm. Scale bars, 10 mm. C, Chemical inhibition of Ras pathway effectors in relation to the expression of angiogenic factors HIF1a and VEGF in T24 bladder cancer cell line that expresses an activating mutation of HRas. Inhibitors and concentrations are noted above the panels (U0126 and PD98059, ERK inhibitors; LY294002, AKT inhibitor; AG490 and S3I-201, STAT3 inhibitors). Note that inhibition of STAT3, but not that of ERK and AKT, led to a significant reduction in HIF1a and VEGF expression.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Proximity Ligation Assay, Inhibition, Activation Assay, Expressing, Cell Culture, Binding Assay, Staining, Produced, Double Staining, Mutagenesis

Journal: Cancer Research

Article Title: PKM2 Is Essential for Bladder Cancer Growth and Maintenance

doi: 10.1158/0008-5472.can-21-0403

Figure Lengend Snippet: Figure 8. Status of glycolysis in relation to PKM2 expression. A, Top, ECAR as analyzed by Seahorse showing glycolysis, gly- colytic capacity, and glycolytic reserve of doxycycline-treated mouse lines L7, L8, L9, and L10 (see Fig. 6 for desig- nation; n ¼ 3 mice/line). The three glycolytic indices were all significantly higher in the L10 mice, which inducibly and markedly overexpressed PKM2 than the L7 WT mice, the L8 mice in which PKM2 was constitutively over- expressed, and the L9 mice, which induciblyand markedlyoverexpressed PKM1. Top, statistical analyses; bot- tom, bar diagram illustration. B, The levels of lactate corresponded well with the glycolytic status, with the highest levels associated with the PKM2-overexpressing L10 mice.

Article Snippet: For the latter, briefly, a full-length mouse Pkm2 cDNA (OriGene) was cloned into a pTRE2pur vector (Addgene).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Proportion of PKM2 expression in cells of different histologic types of benign and malignant ovarian tumor. (B) Representative differential PKM2 expression in benign and malignant groups of epithelial ovarian tumor. * p < 0.001. (C) Proportion of p53 and PKM2 expression in cells of different histologic models of ovarian cancer. (D) Spearman's rho showed moderate correlations between PKM2 and p53 expression. PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: Clinicopathological features.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Kaplan–Meier analysis of the probability of progression-free survival ( p = 0.01) (left) and overall survival ( p = 0.057) (right) in patients with ovarian cancer, stratified according to PKM2 expression ( n = 88). (B) Expression of PKM2 in different ovarian cancer cell lines. (C and D) Time-dependent inhibition of glucose consumption (** p< 0.01,*** p< 0.001) and lactate production in CP70 and SKOV3 cells following shikonin treatment. PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing, Inhibition

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for progression-free and overall survival in patients with ovarian cancer.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) The ECAR according to Seahorse analysis decreased following shikonin treatment in SKOV3 and CP70 cells (* p< 0.05). (B) PKM2 inhibition induced a shift in OCR/ECAR in SKOV3 cells. (C) Dose-dependent inhibition of cancer cell growth in CP70 and SKOV3 cells following shikonin treatment. (D) Migration assay involving CP70 and SKOV3 cells following shikonin treatment. ECAR, extracellular acidification rate; OCR, oxygen-consumption rate; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Inhibition, Migration

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Treatment with shikonin did not cause differences in body weight loss between the treatment and control groups. (B) Representative micro-PET images of xenograft mice in the shikonin-treated group as compared with the control group. (C) Shikonin treatment inhibited tumor formation in vivo following injection of SKOV3 ovarian cancer cells. PET, positron emission tomography; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Control, Micro-PET, In Vivo, Injection, Positron Emission Tomography

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) H&E and IHC stains of mouse tumors following shikonin treatment. (B) Shikonin treatment did not cause apparent pathologic abnormalities in the brain, kidney, liver, or heart according to H&E staining. H&E, hematoxylin and eosin; IHC, immunohistochemistry; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis

doi: 10.1038/s41419-022-04711-1

Figure Lengend Snippet: A RT-PCR analysis on the correlation between YTHDF1 and PKM2 mRNA. B The m6A modification motif of PKM2 mRNA for YTHDF1 binding, analyzed by RMBase V2.0 database ( http://rna.sysu.edu.cn/rmbase/ ). C Breast cancer cell lines were co-transfected with siYTHDF1 and pmirGLO-PKM2 reporter for 48 h to determine the translation efficiency of PKM2 after different treatment, which was calculated by dividing protein yield with mRNA abundance (F-luc/R-luc). D YTHDF1 and PKM2 expression levels in breast cancer cells after the co-transfection with YTHDF1 siRNA and pCDNA3.1-PKM2-3×FLAG. E pH changes in the culture medium of breast cancer cells in different groups after 48 h. F , G The concentration of glucose and lactic acid in the culture medium of the breast cancer cells in different groups after 48 h. H Detection of YTHDF1 and PKM2 expression in the breast cancer cells after co-transfection with pCDNA3.1-YTHDF1-3×FLAG and PKM2 siRNA. I pH changes in the culture medium of breast cancer cells in different groups after 48 h. J , K The concentration of glucose and lactate in the culture medium of the breast cancer cells after 48 h. L Apoptosis levels of breast cancer cells after co-transfection with YTHDF1 siRNA and pCDNA3.1-PKM2-3×FLAG via FACS. M Apoptosis levels of breast cancer cells after co-transfection with pCDNA3.1-YTHDF1-3×FLAG and PKM2 siRNA via FACS. Statistical analysis results are presented as mean ± SEM, student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human YTHDF1 and PKM2 overexpression vectors were constructed by inserting YTHDF1 (NM_017798.4) and PKM2 (NM_002654.6) into pCDNA3.1-3×FLAG (Addgene, #53556) plasmid respectively, of which the sequences were synthesized by Miaolingbio Ptd Ltd.

Techniques: Reverse Transcription Polymerase Chain Reaction, Modification, Binding Assay, Transfection, Expressing, Cotransfection, Concentration Assay

Journal: Cell Death & Disease

Article Title: YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis

doi: 10.1038/s41419-022-04711-1

Figure Lengend Snippet: A , B Impact of YTHDF1 inhibition on the growth of subcutaneous tumors. C Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors at 28 days after transplantation. D , E Impact of YTHDF1 overexpression on the growth of subcutaneous tumors. F Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors. G , H Impact of agomir-16-5p transfection on the growth of subcutaneous tumors. I Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors at 24 days after agomir-16-5p treatment. J – L Immunohistochemical image regarding the lung metastasis of breast tumors in mice after YTHDF1 knockdown, YTHDF1 overexpression or agomir-16-5p treatment. M Immunohistochemical imaging of YTHDF1 and PKM2 in matched normal and cancerous tissues of breast cancer patients. N Schematic illustration showing YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis.

Article Snippet: Human YTHDF1 and PKM2 overexpression vectors were constructed by inserting YTHDF1 (NM_017798.4) and PKM2 (NM_002654.6) into pCDNA3.1-3×FLAG (Addgene, #53556) plasmid respectively, of which the sequences were synthesized by Miaolingbio Ptd Ltd.

Techniques: Inhibition, Western Blot, Expressing, Transplantation Assay, Over Expression, Transfection, Immunohistochemical staining, Knockdown, Imaging