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: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a, Enrichment of GO terms among CDK6-interactors identified in all T-ALL cell lines. p-values, Benjamini-Hochberg-test. b, In vitro kinase reactions using immunoprecipitated endogenous CDK6 and recombinant PFKP or PKM2 ±palbociclib (PALBO). 32P-PFKP/PKM2 denotes phosphorylated proteins, IB, immunoblotting. c, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 2e). d, PFKP and PKM2 activity in cells transfected with empty vector (Vec), D3/CDK6, or kinase-dead CDK6 (D3/CDK6-KD). e, PFKP and PKM2 activity after palbociclib-treatment. Data are mean ±s.d. *P<0.05; **P<0.01 (two-tailed t-test). b,c, representative experiments, out of 2 independent experiments (b), or 3 independent experiments (c, error bars from technical replicates). d,e, n=3 biological replicates. See Supplementary Fig. 1 for gel source data.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: In Vitro, Immunoprecipitation, Recombinant, Western Blot, Phospho-proteomics, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: Flow of glucose-derived carbon into PPP (a), and serine pathway (b) following D3-CDK6 inhibition in T-ALL KOPTK1 cells expressing wild-type PFKP and PKM2 (KOPTK1-WT), or PFKP-S679E and PKM2-S37E mutants (KOPTK1-EE). Levels of NADPH (c), GSH (d), ROS (e) in T-ALL cell lines upon palbociclib-treatment. f, Apoptosis of cells treated with palbociclib and NAC. g, Apoptosis in KOPTK1-WT and KOPTK1-EE cells upon palbociclib-treatment, or following knockdown of CDK6 (h). i,j, In vivo apoptosis of T-ALL cells (in peripheral blood and bone marrow, gated on human CD45+cells) in mice xenografted with KOPTK1-WT or KOPTK1-EE cells. Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, n=4, c-h, n=3, i,j, n=5 biological replicates. See Source data for Fig. 2 for T-ALL xenograft experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Derivative Assay, Inhibition, Expressing, Knockdown, In Vivo, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a,b, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 9i), PFKP and PKM2 activity (c,d), levels of NADPH (e), GSH (f), ROS (g) in palbociclib-treated D3/CDK6-high cells. h, Apoptosis of D3/CDK6-high EBC1 cells expressing wild-type PFKP and PKM2 (EBC1-WT), or PFKP-S679E and PKM2-S37E mutants (EBC1-EE). Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, representative experiments (out of 3 independent experiments, error bars from technical replicates), c-h, n=3 biological replicates.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: PFKP and PKM2 phosphorylation (a,b, from Extended Data Fig. 10i, j), PFKP and PKM2 activity (c,d), GSH (e) and ROS (f) levels in D3/CDK6-high (regressing) and D3/CDK6-low (non-regressing) tumors from ribociclib-treated mice. Data are mean ±s.d. P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). n=3 biological replicates. See Source data for Fig. 5 for PDX drug treatment experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-026-01676-5

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

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

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

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-026-01676-5

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

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

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

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-026-01676-5

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

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

Techniques: Western Blot, Expressing, Staining

Journal: Experimental & Molecular Medicine

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

doi: 10.1038/s12276-026-01676-5

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

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

Techniques: Western Blot, Expressing, Generated, Inhibition