Journal: bioRxiv

Article Title: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma

doi: 10.1101/2021.05.09.443334

Figure Lengend Snippet: A. A schematic showing the mouse Pkm locus, construct targeting Pkm1 -specific exon 9, and the resulting targeted, floxed, and deleted Pkm1 alleles. The KpnI restriction enzyme sites used for Southern blot analysis are marked with “K,” and the new KpnI site introduced by the targeting vector is marked with “K*”. The location of the 5’ probe used for southern blot analysis is also indicated, as are the locations of the genotyping primers (green arrows). B. Southern blot analysis of KpnI-digested genomic DNA from Pkm1 +/+ (+/+), Pkm1 +/fl (f/+), and Pkm1 fl/fl (f/f) mice using the 5’ probe shown in (A). Digestion of genomic DNA harboring the wild-type allele (+) yields an 8.3 kb fragment, while DNA harboring the floxed allele (f) yields a ∼5.0kb fragment. C. Pkm1 mRNA levels in Pkm1 +/+ and Pkm1 -/- anterior prostate tissue as determined by quantitative RT-PCR. Mean+/-SD is shown (n=5). The difference in expression between genotypes is significant (***p<0.001 by Student’s t-test). D. Western blot analysis of Pkm1 , Pkm2 , and vinculin expression in various tissues including the ventral, anterior, and dorsolateral (DLP) prostate lobes that were harvested from wild type and Pkm2 pc-/- mice as indicated.

Article Snippet: Western blots were performed using primary antibodies against Pkm1 (Sigma SAB4200094), Pkm2 (Cell Signaling Technology #4053), Pkm (Cell Signaling Technology #3190; Abcam ab6191), and Vinculin (Sigma, V4505, clone VIN-11-5).

Techniques: Construct, Southern Blot, Plasmid Preparation, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma

doi: 10.1101/2021.05.09.443334

Figure Lengend Snippet: A. Southern blot screening of embryonic stem cell clones for homologous recombination of the Pkm1 targeting construct using the 5’ probe indicated in . Insertion of a novel KpnI site results in a new ∼5.0 kb fragment from the targeted locus following digestion of genomic DNA. Two clones with successful integration are marked with an arrowhead. B. PCR genotyping of genomic DNA from Pkm1 +/+ (+/+), Pkm1 fl/+ (f/+), and Pkm1 fl/fl (f/f) mice as indicated. Genotyping primers anneal at sites indicated in to produce amplicons of 509 bp from the Pkm wild-type allele ( Pkm + ) and 577 bp from the conditional Pkm allele with flanking LoxP sites ( Pkm1 fl ).

Article Snippet: Western blots were performed using primary antibodies against Pkm1 (Sigma SAB4200094), Pkm2 (Cell Signaling Technology #4053), Pkm (Cell Signaling Technology #3190; Abcam ab6191), and Vinculin (Sigma, V4505, clone VIN-11-5).

Techniques: Southern Blot, Clone Assay, Homologous Recombination, Construct

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: 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: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: CSFV infection upregulated PKM2 expression. ( A ) Immunohistochemical analysis of PKM2 expression in normal and CSFV-infected tissues. ( B ) RT-qPCR analysis of PKM2 gene expression in normal and CSFV-infected tissues. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; *** P < 0.001; **** P < 0.0001, and ns, P > 0.05 (one-way ANOVA). ( C ) RT-qPCR analysis of PKM2 gene transcription in CSFV-infected PK-15 and 3D4/2 cells. Error bars indicate the mean (±SD) of three independent experiments. n = 3. * P < 0.05; ** P < 0.01; ***P < 0.001; and ns, P > 0.05 (one-way ANOVA). ( D ) Western blot analysis of PKM2 protein expression in CSFV-infected PK-15 and 3D4/2 cells. The level of protein was quantified using Image-Pro Plus 6.0 software, and the ratios were calculated relative to the tubulin control. Error bars indicate the mean (±SD) of three independent experiments. **** P < 0.0001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Infection, Expressing, Immunohistochemical staining, Quantitative RT-PCR, Gene Expression, Western Blot, Software, Control

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: CSFV affects pyruvate metabolism in PK-15 and 34D/2 cells through PKM2. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were infected with CSFV at an MOI of 1.0 and pyruvate content was measured by spectrophotometric assays in cultured cells at 24 and 48 h. ( C ) PK-15 cells were transfected with PKM2 and then infected with CSFV at an MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. ( D ) 3D4/2 cells were transfected with PKM2 and then infected with CSFV at MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. All measurements were made in triplicates. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Infection, Cell Culture, Transfection

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: Validation of the interaction of PKM2 with NS4A and NS5A by mass spectrometry and immunoprecipitation. ( A ) Mass spectrometry screening of host-protein networks interacting with CSFV NS4A. ( B ) Analysis of the CSFV NS4A protein-host protein interaction network. ( C ) Statistical analysis of host proteins related to metabolism based on MCC using the cytoHubba plugin. ( D ) Statistical analysis of host proteins related to metabolism by setting continuous gradients based on degree values. ( E ) Co-immunoprecipitation analysis of 3×Flag-tagged PKM2 and GFP-tagged NS4A and NS5A by the anti-flag monoclonal antibody (mAb) or by the anti-GFP mAb. PK-15 and 3D4/2 cells were co-transfected with the indicated plasmids (+) or empty vectors (−) for 24 h. The transfected cells were lysed and incubated with a mouse anti-Flag mAb or anti-GFP mAb, followed by incubation with the protein G-agarose for 6 h at 4°C. The immunoprecipitate was analyzed by western blot using the anti-Flag and anti-GFP.

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Biomarker Discovery, Mass Spectrometry, Immunoprecipitation, Transfection, Incubation, Western Blot

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 colocalized with CSFV NS4A and NS5A and promoted the expression of NS4A and NS5A ( A and B ). PK-15 ( A ) and 3D4/2 ( B ) cells were co-transfected with 3× Flag-tagged PKM2 and GFP-tagged NS4A/NS5A. Cells were fixed at 24 h post-transfection and subjected to indirect immunofluorescence assay to detect GFP-NS4A/NS5A (green) and 3× Flag-PKM2 (red) with mouse anti-Flag and rabbit anti-GFP antibodies. The merged image indicates the nucleus by 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining. ( C ) Western blot detection of NS4A protein expression levels in PK-15 cells overexpressing or silencing PKM2. ( D ) Western blot detection of NS5A protein expression levels in PK-15 cells overexpressing or silencing PKM2.

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Expressing, Transfection, Immunofluorescence, Staining, Western Blot

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 positively regulated the proliferation of CSFV in PK-15 and 3D4/2 cells ( A and D ). PK-15 and 3D4/2 cells were transduced with p3×Flag-PKM2 or p3×Flag-CMV ( A ) (siNC or siPKM2) ( D ), followed by infection with CSFV at an MOI of 1.0 or mock infected. Cell samples were analyzed by western blot with antibodies against PKM2, CSFV E2, and tubulin (loading control). ( B and E ) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( C ) siRNA knockdown of PKM2 in PK-15 and 3D4/2 cells transfected with siNC or PKM2 siRNA-1/-2/-3. The expression of PKM2 was assessed by western blot and RT-qPCR at 24 h. Error bars indicate the mean (±SD) of three independent experiments. *** P < 0.001 and **** P < 0.0001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Transduction, Infection, Western Blot, Control, Virus, Knockdown, Transfection, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: Rescue effect of pyruvate on silencing PKM2-induced inhibition of CSFV replication. ( A ) PK-15 and 3D4/2 cells were cultured in a pyruvate-free medium for a while after the addition of pyruvate solution, respectively, followed by infection with CSFV at an MOI of 1.0, and CSFV N pro protein expression was detected after 48 h. ( B ) The relative expression level of NS5B gene in PK-15 and 3D4/2 cells was analyzed by RT-qPCR. Cells were treated as in panel A . ( C ) PK-15 and 3D4/2 cells were treated with pyruvate (5 mM) after being transfected with siPKM2 and then infected with CSFV at an MOI of 1.0. At 48 hpi, cell samples were analyzed by western blot with antibodies against CSFV E2 and tubulin (loading control). ( D ) PK-15 and 3D4/2 cells were treated as in panel C . NS5B gene levels in PK-15 and 3D4/2 cells were assessed using RT-qPCR. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated as in panel C . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and ** P < 0.01 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Inhibition, Cell Culture, Infection, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Virus

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 increased ROS in cells and disrupted mitochondrial function. ( A ) PKM2 overexpression and CSFV infection increased ROS in PK-15 cells. Rosup as a positive control. ( B and C ) The Mitochondrial Membrane Potential Assay Kit (JC-1) detected mitochondrial membrane potential in PK-15 ( B ) and 3D4/2 cells ( C ). In normal mitochondria, JC-1 is present as a polymer with bright red fluorescence and very weak green fluorescence in the cell. When the mitochondrial membrane potential is reduced by treatment with carbonyl cyanide m-chlorobenzyl hydrazone (CCCP), JC-1 cannot be present as a polymer in the mitochondrial matrix, and the intensity of red fluorescence in the mitochondria is significantly reduced. In contrast, green fluorescence in the cytoplasm is enhanced considerably. Image-Pro Plus 6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Over Expression, Infection, Positive Control, Membrane, Polymer, Fluorescence, Software

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 induced mitochondrial fission and mitophagy. ( A ) Confocal microscopy images showing mitochondrial fragmentation in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. Cells have stained the mitochondria with MitoTracker (red) and the cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). In the zoomed images, typical tubular mitochondria in control cells and fragmented mitochondria in PKM2-overexpressed cells are shown. The bar graph represents the average number of mitochondria (red dots) in each cell. Results represent the mean of at least three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001. ( B ) Confocal microscope image showing co-localization of PKM2 with TOM20. Cells were prepared as in panel A . At 24 h, cells were immunostained with the TOM20 antibody (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Image-Pro Plus6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times). ** P < 0.01 and *** P < 0.001 (one-way ANOVA). ( C and D ) Western blot detected the relative expression of the autophagy-associated proteins ATG5, LC3, and p62 in cells with PKM2 overexpression ( C ) and inhibition ( D ). The marker proteins TOM20, VDACI, COXIV, LAMP1, and tubulin (loading control) of mitophagy were also detected by western blot. The level of protein and fluorescence intensity were quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Confocal Microscopy, Transfection, Staining, Control, Microscopy, Software, Fluorescence, Western Blot, Expressing, Over Expression, Inhibition, Marker

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 induced complete mitophagy. ( A and B ) Overexpression of PKM2 increased mitochondrial autophagic vesicle formation in PK-15 ( A ) and 3D4/2 cells ( B ). TEM images revealed the mitochondrial ultrastructure in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were mock handled or PKM2 overexpressed for 24 h and analyzed by TEM. Typical elongated tubular mitochondria in mock cells and fragmented elliptic mitochondria engulfed with membrane-like vesicles in PKM2-overexpressed cells were observed in the zoomed images. Scale bar: 2 µm. ( C ) Quantification of the mitophagosome-like vesicles per cell image (mean ± SD; n ≥ 5 cells; **** P < 0.0001) (one-way ANOVA). ( D and E ) PK-15 ( D ) and 3D4/2 cells ( E ) transiently expressing Mito-mRFP-EGFP were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Over Expression, Membrane, Expressing, Transfection, Fluorescence, Software

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: Silencing PKM2 inhibited mitophagy induced by CSFV. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were transfected with the siRNA of siPKM2 or siNC for 24 h, then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins ATG5, P62, LC3, COXIV, TOM20, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01, and *** P < 0.001 (one-way ANOVA). ( C and D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were transfected with siPKM2 or infected with CSFV for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Transfection, Infection, Western Blot, Expressing, Control, Software, Fluorescence

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: The AMPK-mTOR pathway was upregulated in PKM2-inhibited cells with CSFV infection. ( A and B ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and ( A ) siPKM2 or siNC ( B ). Cell samples were analyzed at 24 and 48 h by immunoblotting with antibodies against AMPK, p-AMPK, p-mTOR, Flag, PKM2, and tubulin (loading control). ( C and D ) PK-15 and 3D4/2 cells were treated with 10 µM Compound C ( C ) or 10 µM AICAR ( D ). DMSO treatment was used as a control. Cell samples were analyzed at 24 h by immunoblotting with antibodies against AMPK and tubulin (loading control). Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; *** P < 0.001, and **** P < 0.0001 (two-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated with DMSO or Compound C and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, p-mTOR, and tubulin (loading control). ( F ) PK-15 and 3D4/2 cells were treated with 10 µM DMSO or 10 µM AICAR and then transfected with siPKM2 or siNC. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, p-mTOR, and tubulin (loading control). ( G ) PK-15 and 3D4/2 cells were transfected with the siPKM2 or siNC for 24 h and then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, and TUBA (loading control).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Infection, Transfection, Western Blot, Control, Expressing

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 activated AMPK and enriched it in mitochondria. ( A ) The effect of FNZ (10 µM) on cell viability of PK-15 and 3D4/2 cells. Cells were treated with different concentrations of FNZ and evaluated by the CCK-8 assay. Error bars represent the mean ± SD; n = 3; ** P < 0.01; *** P < 0.001; **** P < 0.0001; and ns, P > 0.05. ( B ) PK-15 and 3D4/2 cells were treated with FNZ at different times. Western blot was used to analyze the relative expression of VDACI, COXIV, TOM20, HSP60, and tubulin (loading control). ( C ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and then treated with 10 µM FNZ, the same volume of DMSO. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). ( D ) Effect of PKM2 on AMPK mitochondrial translocation. PK-15 cells were transfected with P3×Flag-CMV or Flag-PKM2, followed by mitochondrial isolation using the Mitochondrial Isolation Kit. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: CCK-8 Assay, Western Blot, Expressing, Control, Transfection, Translocation Assay, Isolation, Software

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 regulated mitophagy through the AMPK-mTOR pathway. ( A ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, P62, COXIV, TOM20, LC3, and tubulin (loading control). ( B ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, P62, COXIV, TOM20, LC3, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/Compound C/Mdivi-1 (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. ( D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/AICAR/CCCP (10 µM) for 2 h and then transfected with siNC or siPKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. ns, P > 0.05; *** P < 0.001; and **** P < 0.0001 (two-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Transfection, Western Blot, Control, Software, Expressing, Fluorescence

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: PKM2 promoted CSFV proliferation via AMPK. (A and B) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM AICAR ( A ) or Compound C ( B ), the same volume of DMSO. The relative expression of NS5B mRNA was detected by qRT-PCR at 24 h. ( C and D ) Cell treatment is the same as panel A. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). ( E ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, E2, and tubulin (loading control). ( F ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR (10 µM) for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, E2, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( G ) Cell treatment is the same as panel E . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. ( H ) Cell treatment is the same as panel F . CSFV virus titers in the supernatant were determined as TCID50/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection, Software, Virus

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: Overexpression of PKM2 promoted CSFV proliferation via mitophagy. ( A and B ) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM Mdivi-1, the same volume of DMSO. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C and D ) Cell treatment is the same as panel A. CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars represent the mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, P > 0.05 (one-way ANOVA).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Over Expression, Infection, Western Blot, Control, Software, Virus

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: The effect of RNA interference on cell viability. The cell viability of PK-15 (A) cells and 3D4/2 (B) cells transfected with siNC or PKM2 siRNA-1/-2/-3 was analyzed using the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; ns, P > 0.05).

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Transfection, CCK-8 Assay

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: A model for PKM2 promoted CSFV proliferation by inducing mitophagy via the AMPK-mTOR signaling pathway. Image created with biorender.com.

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques:

Journal: Journal of Virology

Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

doi: 10.1128/jvi.01751-23

Figure Lengend Snippet: Primers used in this study

Article Snippet: The primary antibodies used in the study were specific for LC3B (Cell Signaling Technology, 2775), ATG5 (Beyotim, AF2269), BECN1 (Cell Signaling Technology, 3495), LAMP1 (Beyotim, AF7353), VDACI (Beyotim, AF1027), COXIV (Beyotim, AF6549), TOM20 (Beyotim, AF1717), SQSTM1/p62 (Cell Signaling Technology, 23214), HSP60 (Beyotim, AF1771), AMPK (Beyotim, AF1627), p-AMPK (Beyotim, AA393), p-mTOR (Beyotim, AF5869), PKM2 (Santa Cruz Biotechnology, sc-365684), GAPDH (Beyotime, AG019), Tubulin (Beyotime, AT819), mouse monoclonal anti-Flag (Sigma-Aldrich, F7425), and Mouse anti-GFP antibody (AG281).

Techniques: Sequencing