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  • 99
    Thermo Fisher actin beta β actin
    GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and <t>β-actin,</t> Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.
    Actin Beta β Actin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 36 article reviews
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    99
    Millipore ϐ actin
    GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and <t>β-actin,</t> Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.
    ϐ Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ϐ actin/product/Millipore
    Average 99 stars, based on 62 article reviews
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    95
    Millipore β actin
    HIF-1α silencing in PHD2-deficient chondrocytes ( a ) Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α) (n=3 biologically independent samples). ( b ) HIF-1α and Lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( c-f ) Oxygen consumption ( c ), glycolytic flux ( d ), energy charge ( e ) and energy status ( f ) of cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( g ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( h-i ) Proliferation ( h ) and collagen synthesis ( i ) in cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( j-k ) BiP ( j ), cleaved (c)ATF6 ( k ) and <t>β-actin</t> immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( l-m ) Hydroxyproline (OH-Pro) ( l ; n=6 biologically independent samples) and α-ketoglutarate (αKG) levels ( m ; n=5 biologically independent samples) in cultured chondrocytes, transduced with shScr or shHIF-1α. Data are means ± SEM. # p
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 67346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Millipore
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    94
    Cell Signaling Technology Inc beta actin
    S100A10 is down-regulated by infection of Lv-miR-590-5P. ( A ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P. After 96 h, the cells were collected and total RNA was extracted and subjected to quantitative PCR. U6 was used as an internal standard; ( B ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P, and after 96 hours, the cells were collected and total protein was extracted and subjected to western blotting. For western blotting, cell lysates were separated on a 14% (S100A10) or 13% <t>(beta-actin)</t> SDS polyacrylamidegel, and the blots were detected with a specific anti-S100A10 antibody, with beta-actin as an internal standard **, t -test significant at p
    Beta Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta actin/product/Cell Signaling Technology Inc
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    85
    Cell Signaling Technology Inc beta β actin
    S100A10 is down-regulated by infection of Lv-miR-590-5P. ( A ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P. After 96 h, the cells were collected and total RNA was extracted and subjected to quantitative PCR. U6 was used as an internal standard; ( B ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P, and after 96 hours, the cells were collected and total protein was extracted and subjected to western blotting. For western blotting, cell lysates were separated on a 14% (S100A10) or 13% <t>(beta-actin)</t> SDS polyacrylamidegel, and the blots were detected with a specific anti-S100A10 antibody, with beta-actin as an internal standard **, t -test significant at p
    Beta β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta β actin/product/Cell Signaling Technology Inc
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    90
    Santa Cruz Biotechnology beta actin β actin
    S100A10 is down-regulated by infection of Lv-miR-590-5P. ( A ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P. After 96 h, the cells were collected and total RNA was extracted and subjected to quantitative PCR. U6 was used as an internal standard; ( B ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P, and after 96 hours, the cells were collected and total protein was extracted and subjected to western blotting. For western blotting, cell lysates were separated on a 14% (S100A10) or 13% <t>(beta-actin)</t> SDS polyacrylamidegel, and the blots were detected with a specific anti-S100A10 antibody, with beta-actin as an internal standard **, t -test significant at p
    Beta Actin β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta actin β actin/product/Santa Cruz Biotechnology
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    99
    Boster Bio antibody against β actin β actin
    NT-Sr promote RANKL-induced phosphorylation of ERK and inhibit the Akt/NFATc1 pathway. RAW264.7 cells were cultured on different samples for 3 d and stimulated with or without 100 ng/mL RANKL for 30 min, and the total protein was then collected for immunoblot analysis. ( a ) RANKL-induced phosphorylation of ERK, ( b ) p-Akt and NFATc1 were detected using <t>β-actin,</t> total ERK and Akt as loading controls. ( c ) A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. Full-length blots are presented in Supplementary Figure 4 . The numbers 1, 2, 3 and 4 in the figure represent Ti, TiO 2 -NTs, NT-Sr1h and NT-Sr3h, respectively. * , **p
    Antibody Against β Actin β Actin, supplied by Boster Bio, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    WuXi AppTec β actin
    Extracellular AGR2 enhances RhoA, CDC42 expression and stimulates FAK phosphorylation in NIH3T3 cells. (a) Western blot analysis of RhoA expression in NIH3T3 cells stimulated for 24 h with AGR2 coupled with or without bFGF. The concentration of AGR2 is 500 ng/ml. The concentration of bFGF is 1 ng/ml. (b) NIH3T3 cells were treated by 500 ng/ml AGR2 for the indicated time. RhoA, cyclin D1, P21, ERK1/2, pERK1/2, CDC42, Rac1, RhoB, and RhoC were detected by western blots. <t>β-actin</t> served as loading control. (c) Significant reduction in RhoA expression, cyclin D1 expression, and FAK phosphorylation was observed in the NIH3T3 cells pretreated with 20 nM/ml FGFR1 inhibitor PD173074 and 10 nM/ml VEGFR inhibitor axitinib before AGR2 treatment. β-actin served as loading control.
    β Actin, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 94/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and β-actin, Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.

    Journal: SpringerPlus

    Article Title: HER-2 gene amplification in human breast cancer without concurrent HER-2 over-expression

    doi: 10.1186/2193-1801-2-386

    Figure Lengend Snippet: GRB7 and HER-2 expression in frozen breast tumor tissues. a GRB7 and HER-2 Western Blot Analysis. Densitometric analysis is presented in Additional file 1 : Table S1. b HER-2 IHC testing of HER-2 FISH positive tumors which do not over-express HER-2 protein by Western blot analysis. Sample 012 is positive control with 3+ HER-2 over-expression; sample 002 is negative control with 0 HER-2 expression. Magnification bars represent 50 micron. c GRB7 and HER2 mRNA expression by multiplex RT-PCR. Top panel: GRB7 and β-actin, Bottom panel: HER-2 and β-actin. Amplified: positive control with HER-2 and GRB 7 over-expression. Non-amplified: negative control - without either HER-2 or GRB7 over-expression. H 2 O: negative control without RT product. X: 4 tumors over-expressing GRB7 but not HER-2. O: 1 tumor over-expressing both GRB7 and HER-2.

    Article Snippet: The ProtoScript MuLV Taq RTPCR kit was used for the detection of GRB7 and HER-2 mRNAs from a single first strand cDNA synthesis followed by PCR amplification with HER-2 or GRB7 specific primers and β- actin as internal control (Invitrogen), respectively.

    Techniques: Expressing, Western Blot, Immunohistochemistry, Fluorescence In Situ Hybridization, Positive Control, Over Expression, Negative Control, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    HIF-1α silencing in PHD2-deficient chondrocytes ( a ) Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α) (n=3 biologically independent samples). ( b ) HIF-1α and Lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( c-f ) Oxygen consumption ( c ), glycolytic flux ( d ), energy charge ( e ) and energy status ( f ) of cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( g ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( h-i ) Proliferation ( h ) and collagen synthesis ( i ) in cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( j-k ) BiP ( j ), cleaved (c)ATF6 ( k ) and β-actin immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( l-m ) Hydroxyproline (OH-Pro) ( l ; n=6 biologically independent samples) and α-ketoglutarate (αKG) levels ( m ; n=5 biologically independent samples) in cultured chondrocytes, transduced with shScr or shHIF-1α. Data are means ± SEM. # p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: HIF-1α silencing in PHD2-deficient chondrocytes ( a ) Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α) (n=3 biologically independent samples). ( b ) HIF-1α and Lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( c-f ) Oxygen consumption ( c ), glycolytic flux ( d ), energy charge ( e ) and energy status ( f ) of cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( g ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( h-i ) Proliferation ( h ) and collagen synthesis ( i ) in cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( j-k ) BiP ( j ), cleaved (c)ATF6 ( k ) and β-actin immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( l-m ) Hydroxyproline (OH-Pro) ( l ; n=6 biologically independent samples) and α-ketoglutarate (αKG) levels ( m ; n=5 biologically independent samples) in cultured chondrocytes, transduced with shScr or shHIF-1α. Data are means ± SEM. # p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Expressing, Cell Culture, Transduction, shRNA

    Metabolic alterations in PHD2-deficient chondrocytes ( a ) Rhodamine labelling of mitochondria, with quantification of mitochondrial content (n= samples from 3 Phd2 chon+ and 4 Phd2 chon- mice). Yellow line denotes cell membrane. ( b-c ) Immunoblot of C-MYC ( b ), LC3-II ( c ) and β-actin levels in cultured chondrocytes. Representative images of 4 independent experiments are shown. ( d ) Oxygen consumption in cultured chondrocytes (n=9 biologically independent samples). ( e ) Pimonidazole immunostaining on neonatal (P2.5) growth plates, with a higher magnification of the boxed area and quantification of pimonidazole-positive cells within the growth plate (n=4 mice). ( f ) Glucose oxidation (GO), fatty acid oxidation (FAO) and glutamine oxidation (QO) in cultured chondrocytes (n=6 biologically independent samples). ( g ) Glucose (Glc) uptake and lactate (Lac) secretion (n=6 biologically independent samples). ( h ) Glycolytic flux (n=6 biologically independent samples). ( i ) Fractional contribution of 13 C 6 - Glc to Lac, citrate (Cit), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum) and malate (Mal) (n=6 biologically independent samples). ( j-l ) ATP content ( j ), energy charge ([ATP] + ½ [ADP] / [ATP] + [ADP] + [AMP]; k ), and energy status (ratio of ATP to AMP levels; l ) (n=6 biologically independent samples). ( m ) Apoptosis rate of cultured chondrocytes (n=4 independent experiments). ( n ) TUNEL immunostaining of neonatal growth plates (n=6 mice). ( o ) ATP production resulting from glycolysis, GO, FAO and QO in cultured chondrocytes (n=6 biologically independent samples. ( p ) Proliferation rate of cultured chondrocytes (n=4 independent experiments). ( q ) Immunoblot of Na + /K + ATPase and β-actin levels. Representative images of 3 independent experiments are shown. ( r ) Normalized Ca 2+ -rise in the cytosol of cultured chondrocytes upon stimulation with thapsigargin (TG) in the presence of EGTA (n=4 biologically independent samples). ( s ) Quantification of the Ca 2+ release from the endoplasmic reticulum (ER) upon stimulation with TG (n=4 biologically independent samples). ( t ) 45 Ca 2+ loading capacity of the ER of permeabilised chondrocytes in intracellular-like medium supplemented with 5 mM Mg/ATP and 45 Ca 2+ (n=4 biologically independent samples). ( u-v ) Total protein ( u ) and proteoglycan synthesis ( v ) (n=8 biologically independent samples). Data are means ± SEM in ( a-d, f-m, o-v ), or means ± SD in ( e, n ). *p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Metabolic alterations in PHD2-deficient chondrocytes ( a ) Rhodamine labelling of mitochondria, with quantification of mitochondrial content (n= samples from 3 Phd2 chon+ and 4 Phd2 chon- mice). Yellow line denotes cell membrane. ( b-c ) Immunoblot of C-MYC ( b ), LC3-II ( c ) and β-actin levels in cultured chondrocytes. Representative images of 4 independent experiments are shown. ( d ) Oxygen consumption in cultured chondrocytes (n=9 biologically independent samples). ( e ) Pimonidazole immunostaining on neonatal (P2.5) growth plates, with a higher magnification of the boxed area and quantification of pimonidazole-positive cells within the growth plate (n=4 mice). ( f ) Glucose oxidation (GO), fatty acid oxidation (FAO) and glutamine oxidation (QO) in cultured chondrocytes (n=6 biologically independent samples). ( g ) Glucose (Glc) uptake and lactate (Lac) secretion (n=6 biologically independent samples). ( h ) Glycolytic flux (n=6 biologically independent samples). ( i ) Fractional contribution of 13 C 6 - Glc to Lac, citrate (Cit), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum) and malate (Mal) (n=6 biologically independent samples). ( j-l ) ATP content ( j ), energy charge ([ATP] + ½ [ADP] / [ATP] + [ADP] + [AMP]; k ), and energy status (ratio of ATP to AMP levels; l ) (n=6 biologically independent samples). ( m ) Apoptosis rate of cultured chondrocytes (n=4 independent experiments). ( n ) TUNEL immunostaining of neonatal growth plates (n=6 mice). ( o ) ATP production resulting from glycolysis, GO, FAO and QO in cultured chondrocytes (n=6 biologically independent samples. ( p ) Proliferation rate of cultured chondrocytes (n=4 independent experiments). ( q ) Immunoblot of Na + /K + ATPase and β-actin levels. Representative images of 3 independent experiments are shown. ( r ) Normalized Ca 2+ -rise in the cytosol of cultured chondrocytes upon stimulation with thapsigargin (TG) in the presence of EGTA (n=4 biologically independent samples). ( s ) Quantification of the Ca 2+ release from the endoplasmic reticulum (ER) upon stimulation with TG (n=4 biologically independent samples). ( t ) 45 Ca 2+ loading capacity of the ER of permeabilised chondrocytes in intracellular-like medium supplemented with 5 mM Mg/ATP and 45 Ca 2+ (n=4 biologically independent samples). ( u-v ) Total protein ( u ) and proteoglycan synthesis ( v ) (n=8 biologically independent samples). Data are means ± SEM in ( a-d, f-m, o-v ), or means ± SD in ( e, n ). *p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Mouse Assay, Cell Culture, Immunostaining, TUNEL Assay

    Administration of α-ketoglutarate increases collagen hydroxylation and bone mass in wild-type mice ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without supplementation of dimethyl-αKG (hereafter αKG) (n=4 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with αKG (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without αKG supplementation (n=4 biologically independent samples). ( e ) Immunoblot of BiP, cleaved (c)ATF6 and β-actin in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with αKG (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with αKG, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with αKG, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with αKG, and quantification of trabecular bone volume (TBV) (n=5 mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with αKG (n=3 biologically independent samples). Data are means ± SEM in ( a-b, d-e, j ), or means ± SD in ( c, f-i, k ). # p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Administration of α-ketoglutarate increases collagen hydroxylation and bone mass in wild-type mice ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without supplementation of dimethyl-αKG (hereafter αKG) (n=4 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with αKG (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without αKG supplementation (n=4 biologically independent samples). ( e ) Immunoblot of BiP, cleaved (c)ATF6 and β-actin in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with αKG (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with αKG, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with αKG, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with αKG, and quantification of trabecular bone volume (TBV) (n=5 mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with αKG (n=3 biologically independent samples). Data are means ± SEM in ( a-b, d-e, j ), or means ± SD in ( c, f-i, k ). # p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Mouse Assay, Cell Culture, Staining, Immunostaining, Derivative Assay

    Phenotype of Phd2 chon- mice ( a ) Southern blot analysis showing efficient and selective recombination (black arrowhead) of the Phd2 gene in neonatal (P2.5) growth plate tissue from Phd2 chon- mice. Representative images of 3 independent experiments are shown. ( b ) Phd1 , Phd2 and Phd3 mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( c-d ) Immunoblot of PHD2 and β-actin ( c ), and of HIF-1α, HIF-2α and Lamin A/C ( d ) levels in growth plate tissue ( c ) and cultured chondrocytes ( d ). Representative images of 4 independent experiments are shown. ( e ) Quantification of tibia length (n=11 Phd2 chon+ − 8 Phd2 chon- mice), body weight (n=11 Phd2 chon+ − 8 Phd2 chon- mice), lean body mass (n=4 mice) and fat mass (n=4 mice) of adult (14-week-old) mice. ( f ) Growth-related phenotype in adult Phd2 chon- mice. ( g ) Sox9 , Col2 , Col10 , Pthrp , Ihh , Mmp9 , Mmp13 and Opn mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( h ) In situ hybridization for Col2 , Col10 , Pthrp and Ihh on neonatal growth plates (n=4 biologically independent samples; scale bar is 250 μm). ( i ) Quantification of trabecular number (Tb.N; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice) and thickness (Tb.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), cortical thickness (Ct.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), calvarial thickness (calv.Th; n=4 mice) and porosity (calv.Po; n=4 mice) in neonatal and adult mice. Nd is not determined. ( j ) Quantification of osteoblast number (N.Ob/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice), osteoblast surface (Ob.S/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice) and osteoid surface per bone surface (O.S/B.S; n=6 mice), bone formation rate (BFR; n=4 mice), mineral apposition rate (MAR; n=4 mice), osteoclast surface per bone surface (Oc.S/B.S; n=11 Phd2 chon+ − 7 Phd2 chon- mice), blood vessel number per tissue surface (N.BV/T.S; ; n=9 Phd2 chon+ − 6 Phd2 chon- mice), and serum osteocalcin (OCN; n=16 biologically independent samples), CTx-I (n=8 biologically independent samples) and CTx-II levels (n=9 biologically independent samples) in adult mice. ( k-l ) Representative images of TRAP-positive multinuclear cells formed after one week of culture ( k ) with quantification ( l ) of the number of osteoclasts formed per well (n=4 biologically independent samples; scale bar is 50 μm). Quantification was based on the number of nuclei per osteoclast. ( m ) Type I collagen (COL1) and COL2 immunostaining of the metaphysis of neonatal mice (n=8 mice). Scale bar is 100 μm. ( n ) Quantification of the cell/extracellular matrix (ECM) ratio in two zones of the growth plate (n=8 mice). Data are means ± SEM in ( c, d, l ), or means ± SD in ( b, e, g, i, j, n ). *p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Phenotype of Phd2 chon- mice ( a ) Southern blot analysis showing efficient and selective recombination (black arrowhead) of the Phd2 gene in neonatal (P2.5) growth plate tissue from Phd2 chon- mice. Representative images of 3 independent experiments are shown. ( b ) Phd1 , Phd2 and Phd3 mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( c-d ) Immunoblot of PHD2 and β-actin ( c ), and of HIF-1α, HIF-2α and Lamin A/C ( d ) levels in growth plate tissue ( c ) and cultured chondrocytes ( d ). Representative images of 4 independent experiments are shown. ( e ) Quantification of tibia length (n=11 Phd2 chon+ − 8 Phd2 chon- mice), body weight (n=11 Phd2 chon+ − 8 Phd2 chon- mice), lean body mass (n=4 mice) and fat mass (n=4 mice) of adult (14-week-old) mice. ( f ) Growth-related phenotype in adult Phd2 chon- mice. ( g ) Sox9 , Col2 , Col10 , Pthrp , Ihh , Mmp9 , Mmp13 and Opn mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( h ) In situ hybridization for Col2 , Col10 , Pthrp and Ihh on neonatal growth plates (n=4 biologically independent samples; scale bar is 250 μm). ( i ) Quantification of trabecular number (Tb.N; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice) and thickness (Tb.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), cortical thickness (Ct.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), calvarial thickness (calv.Th; n=4 mice) and porosity (calv.Po; n=4 mice) in neonatal and adult mice. Nd is not determined. ( j ) Quantification of osteoblast number (N.Ob/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice), osteoblast surface (Ob.S/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice) and osteoid surface per bone surface (O.S/B.S; n=6 mice), bone formation rate (BFR; n=4 mice), mineral apposition rate (MAR; n=4 mice), osteoclast surface per bone surface (Oc.S/B.S; n=11 Phd2 chon+ − 7 Phd2 chon- mice), blood vessel number per tissue surface (N.BV/T.S; ; n=9 Phd2 chon+ − 6 Phd2 chon- mice), and serum osteocalcin (OCN; n=16 biologically independent samples), CTx-I (n=8 biologically independent samples) and CTx-II levels (n=9 biologically independent samples) in adult mice. ( k-l ) Representative images of TRAP-positive multinuclear cells formed after one week of culture ( k ) with quantification ( l ) of the number of osteoclasts formed per well (n=4 biologically independent samples; scale bar is 50 μm). Quantification was based on the number of nuclei per osteoclast. ( m ) Type I collagen (COL1) and COL2 immunostaining of the metaphysis of neonatal mice (n=8 mice). Scale bar is 100 μm. ( n ) Quantification of the cell/extracellular matrix (ECM) ratio in two zones of the growth plate (n=8 mice). Data are means ± SEM in ( c, d, l ), or means ± SD in ( b, e, g, i, j, n ). *p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Mouse Assay, Southern Blot, Cell Culture, In Situ Hybridization, Immunostaining

    PHD2-deficient chondrocytes display enhanced glutamine metabolism ( a ) Intracellular glutamate (Glu), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum), malate (Mal) and citrate (Cit) levels in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( b ) Ratio of αKG/Suc and αKG/Fum (n=3 biologically independent samples). ( c ) GLS1 and β-actin immunoblot of cultured chondrocytes transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α). Representative images of 3 independent experiments are shown. ( d ) Immunoblot of GLS1, GLS2 and β-actin in cultured chondrocytes, compared to HeLa cells. Representative images of 3 independent experiments are shown. ( e ) Fractional contribution of 13 C 5 -glutamine (Gln) to Glu, αKG, Suc, Fum, Mal and Cit in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( f ) Citrate mass isotopomer distribution (MID) from 13 C 5 -Gln (n=3 biologically independent samples). ( g ) Relative abundance of reductive carboxylation-specific mass isotopomers of Cit, Mal and Fum (n=3 biologically independent samples). ( h ) Type II collagen (COL2) immunostaining of the tibia of neonatal (P2.5) mice treated with BPTES and/or αKG with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with BPTES, with or without αKG, and quantification of trabecular bone volume (TBV) (n=6 for Phd2 chon+ -veh, Phd2 chon+ -BPTES+αKG or Phd2 chon- -veh mice; n=5 for Phd2 chon+ -BPTES mice; and n=7 for Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with BPTES, with or without αKG (n=3 biologically independent samples). ( l ) Immunoblot of p-AMPK T172 and AMPK in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( m ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, treated with BPTES, with or without αKG (n=3 biologically independent samples). ( n ) Tibia length of mice treated with BPTES, with or without αKG (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( o ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. Data are means ± SEM in ( a-g, j, l, m, o ), or means ± SD in ( h, i, k, n ). *p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: PHD2-deficient chondrocytes display enhanced glutamine metabolism ( a ) Intracellular glutamate (Glu), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum), malate (Mal) and citrate (Cit) levels in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( b ) Ratio of αKG/Suc and αKG/Fum (n=3 biologically independent samples). ( c ) GLS1 and β-actin immunoblot of cultured chondrocytes transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α). Representative images of 3 independent experiments are shown. ( d ) Immunoblot of GLS1, GLS2 and β-actin in cultured chondrocytes, compared to HeLa cells. Representative images of 3 independent experiments are shown. ( e ) Fractional contribution of 13 C 5 -glutamine (Gln) to Glu, αKG, Suc, Fum, Mal and Cit in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( f ) Citrate mass isotopomer distribution (MID) from 13 C 5 -Gln (n=3 biologically independent samples). ( g ) Relative abundance of reductive carboxylation-specific mass isotopomers of Cit, Mal and Fum (n=3 biologically independent samples). ( h ) Type II collagen (COL2) immunostaining of the tibia of neonatal (P2.5) mice treated with BPTES and/or αKG with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with BPTES, with or without αKG, and quantification of trabecular bone volume (TBV) (n=6 for Phd2 chon+ -veh, Phd2 chon+ -BPTES+αKG or Phd2 chon- -veh mice; n=5 for Phd2 chon+ -BPTES mice; and n=7 for Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with BPTES, with or without αKG (n=3 biologically independent samples). ( l ) Immunoblot of p-AMPK T172 and AMPK in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( m ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, treated with BPTES, with or without αKG (n=3 biologically independent samples). ( n ) Tibia length of mice treated with BPTES, with or without αKG (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( o ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. Data are means ± SEM in ( a-g, j, l, m, o ), or means ± SD in ( h, i, k, n ). *p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Cell Culture, Transduction, shRNA, Immunostaining, Mouse Assay, Derivative Assay, BrdU Incorporation Assay

    Inhibition of pyruvate uptake does not affect collagen or bone properties ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i) (n=3 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with MCT2i (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment (n=4 biologically independent samples). ( e ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with MCT2i (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume (TBV) (n=5 mice). Data are means ± SEM in ( a-b, d-e ), or means ± SD in ( c, f-i ). # p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Inhibition of pyruvate uptake does not affect collagen or bone properties ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i) (n=3 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with MCT2i (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment (n=4 biologically independent samples). ( e ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with MCT2i (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume (TBV) (n=5 mice). Data are means ± SEM in ( a-b, d-e ), or means ± SD in ( c, f-i ). # p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Inhibition, Cell Culture, Mouse Assay, Staining, Immunostaining

    Genetic confirmation of HIF-1α signalling and metabolic adaptations ( a-c ) Immunoblot of HIF-1α ( a ), GLS1 ( b ), PDK1 ( c ), Lamin A/C and β-actin in cultured control or PHD2-deficient (PHD2 KD ) periosteal cells, transduced with scrambled shRNA (shScr; -) or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( d ) Toluidine Blue staining of bone ossicles (n=5 biologically independent samples). Arrowheads indicate cartilage remnants (scale bar is 100 μm). ( e ) 3D CT models of bone ossicles, with quantification of the mineralized tissue volume (MV/TV) (n=5 biologically independent samples). ( f-g ) Immunoblot of GLS1 ( f ), PDK1 ( g ), and β-actin in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( h ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( i-k ) Proliferation ( i ), α-ketoglutarate (αKG) levels ( j ) and hydroxyproline (OH-Pro) content ( k ) in cultured chondrocytes, transduced with shScr or gene-specific shRNAs (n=5 biologically independent samples). Data are means ± SEM in ( a-c, f-k ), or means ± SD in ( e ). # p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Genetic confirmation of HIF-1α signalling and metabolic adaptations ( a-c ) Immunoblot of HIF-1α ( a ), GLS1 ( b ), PDK1 ( c ), Lamin A/C and β-actin in cultured control or PHD2-deficient (PHD2 KD ) periosteal cells, transduced with scrambled shRNA (shScr; -) or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( d ) Toluidine Blue staining of bone ossicles (n=5 biologically independent samples). Arrowheads indicate cartilage remnants (scale bar is 100 μm). ( e ) 3D CT models of bone ossicles, with quantification of the mineralized tissue volume (MV/TV) (n=5 biologically independent samples). ( f-g ) Immunoblot of GLS1 ( f ), PDK1 ( g ), and β-actin in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( h ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( i-k ) Proliferation ( i ), α-ketoglutarate (αKG) levels ( j ) and hydroxyproline (OH-Pro) content ( k ) in cultured chondrocytes, transduced with shScr or gene-specific shRNAs (n=5 biologically independent samples). Data are means ± SEM in ( a-c, f-k ), or means ± SD in ( e ). # p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Cell Culture, Transduction, shRNA, Staining

    mTOR signalling and the unfolded protein response in PHD2-deficient chondrocytes ( a ) Immunoblot and quantification of phosphorylated (at Serine 2448) mTOR (p-mTOR S2448 ), mTOR, phosphorylated (at Threonine 389 and Serine 371) p70 S6 kinase (p-p70 S6K T389 and p-p70 S6K S371 ), p70 S6K, phosphorylated (at Serine 235 and 236) S6 (p-S6 S235/236 ) and S6, phosphorylated (at Threonine 37 and 46) 4E-BP1 (p-4E-BP1 T37/46 ), 4E-BP1 and β-actin in cultured chondrocytes. Representative images of 3 independent experiments are shown. ( b-c ) p-S6 S235/236 immunostaining on neonatal (P2.5) growth plates ( b ), with a higher magnification of the boxed area and quantification ( c ) of the p-S6 + area (n=6 mice). GP is growth plate, PS is primary spongiosa. ( d ) Immunoblot of p-S6 S235/236 and S6 in cultured chondrocytes. Cells were either cultured in full medium or in nutrient-deprived conditions (PBS), and then switched to full medium for indicated times. Representative images of 3 independent experiments are shown. These data show the absence of enhanced mTOR signalling. ( e ) Immunoblot and quantification of BiP, (p-)eIF2α, ATF4 and cleaved (c)ATF6 protein levels. Representative images of 3 independent experiments are shown. ( f ) Spliced Xbp-1 ( Xbp-1s ) mRNA levels in neonatal growth plates (n=8 biologically independent samples). ( g-h ) BiP and cATF6 immunostaining ( g ) of neonatal growth plates with quantification ( h ) of the percentage of positive cells (n=6 mice). Data are means ± SEM in ( a, d, e ), or means ± SD in ( c, f, h ). *p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: mTOR signalling and the unfolded protein response in PHD2-deficient chondrocytes ( a ) Immunoblot and quantification of phosphorylated (at Serine 2448) mTOR (p-mTOR S2448 ), mTOR, phosphorylated (at Threonine 389 and Serine 371) p70 S6 kinase (p-p70 S6K T389 and p-p70 S6K S371 ), p70 S6K, phosphorylated (at Serine 235 and 236) S6 (p-S6 S235/236 ) and S6, phosphorylated (at Threonine 37 and 46) 4E-BP1 (p-4E-BP1 T37/46 ), 4E-BP1 and β-actin in cultured chondrocytes. Representative images of 3 independent experiments are shown. ( b-c ) p-S6 S235/236 immunostaining on neonatal (P2.5) growth plates ( b ), with a higher magnification of the boxed area and quantification ( c ) of the p-S6 + area (n=6 mice). GP is growth plate, PS is primary spongiosa. ( d ) Immunoblot of p-S6 S235/236 and S6 in cultured chondrocytes. Cells were either cultured in full medium or in nutrient-deprived conditions (PBS), and then switched to full medium for indicated times. Representative images of 3 independent experiments are shown. These data show the absence of enhanced mTOR signalling. ( e ) Immunoblot and quantification of BiP, (p-)eIF2α, ATF4 and cleaved (c)ATF6 protein levels. Representative images of 3 independent experiments are shown. ( f ) Spliced Xbp-1 ( Xbp-1s ) mRNA levels in neonatal growth plates (n=8 biologically independent samples). ( g-h ) BiP and cATF6 immunostaining ( g ) of neonatal growth plates with quantification ( h ) of the percentage of positive cells (n=6 mice). Data are means ± SEM in ( a, d, e ), or means ± SD in ( c, f, h ). *p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Cell Culture, Immunostaining, Mouse Assay

    Normalization of glucose oxidation corrects the energy deficit in PHD2-deficient chondrocytes ( a-e ) Glucose oxidation ( a ), oxygen consumption ( b ), palmitate oxidation ( c ), glutamine oxidation ( d ) and glycolytic flux ( e ) in cultured chondrocytes, with or without DCA treatment (n=6 biologically independent samples for a , n=3 biologically independent samples for b-e ). ( f ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, with or without DCA treatment (n=3 biologically independent samples). ( g ) Tibia length of mice treated with DCA (n=5 Phd2 chon+ -veh, Phd2 chon- -veh or Phd2 chon- -DCA mice - n=7 Phd2 chon+ -DCA). ( h-i ) BiP ( h ), cleaved (c)ATF6 ( i ) and β-actin immunoblot in cultured DCA-treated chondrocytes. Representative images of 3 independent experiments are shown. ( j ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with DCA, with or without BPTES (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( k ) Type II collagen (COL2) immunostaining of the tibia of mice treated with DCA, with or without BPTES with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 from Phd2 chon+ -veh mice - n=7 from Phd2 chon- -veh, Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( l ) 3D microCT models of the tibial metaphysis of mice treated with DCA, with or without BPTES, and quantification of trabecular bone volume (TBV) (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( m ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes treated with DCA, with or without BPTES (n=4 biologically independent samples). ( n ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with DCA, with or without BPTES (n=3 biologically independent samples). Data are means ± SEM in ( a-f, h-i, m ), or means ± SD in ( g, j-l, n ). # p

    Journal: Nature

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    doi: 10.1038/s41586-019-0874-3

    Figure Lengend Snippet: Normalization of glucose oxidation corrects the energy deficit in PHD2-deficient chondrocytes ( a-e ) Glucose oxidation ( a ), oxygen consumption ( b ), palmitate oxidation ( c ), glutamine oxidation ( d ) and glycolytic flux ( e ) in cultured chondrocytes, with or without DCA treatment (n=6 biologically independent samples for a , n=3 biologically independent samples for b-e ). ( f ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, with or without DCA treatment (n=3 biologically independent samples). ( g ) Tibia length of mice treated with DCA (n=5 Phd2 chon+ -veh, Phd2 chon- -veh or Phd2 chon- -DCA mice - n=7 Phd2 chon+ -DCA). ( h-i ) BiP ( h ), cleaved (c)ATF6 ( i ) and β-actin immunoblot in cultured DCA-treated chondrocytes. Representative images of 3 independent experiments are shown. ( j ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with DCA, with or without BPTES (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( k ) Type II collagen (COL2) immunostaining of the tibia of mice treated with DCA, with or without BPTES with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 from Phd2 chon+ -veh mice - n=7 from Phd2 chon- -veh, Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( l ) 3D microCT models of the tibial metaphysis of mice treated with DCA, with or without BPTES, and quantification of trabecular bone volume (TBV) (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( m ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes treated with DCA, with or without BPTES (n=4 biologically independent samples). ( n ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with DCA, with or without BPTES (n=3 biologically independent samples). Data are means ± SEM in ( a-f, h-i, m ), or means ± SD in ( g, j-l, n ). # p

    Article Snippet: Membranes were blocked with 5% dry milk or bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 for 60 minutes at room temperature and incubated overnight at 4°C with primary antibodies against PHD2 (Bio-Techne), HIF-1α (Bio-Techne), HIF-2α (Abcam), phosphorylated AMPK (Threonin 172, p-AMPKT172 ; Cell Signaling Technologies), AMPK (Cell Signaling Technologies), Na+ /K+ ATPase (Cell Signaling Technologies), LC3-II (Cell Signaling Technologies), C-MYC (Cell Signaling Technologies) BiP (Santa Cruz Biotechnologies), phosphorylated eIF2α (Cell Signaling Technologies), eIF2α (Cell Signaling Technologies), ATF4 (Santa Cruz Biotechnologies), ATF6 (Bio-Techne), COL2 (Merck), glutaminase 1 (GLS1; Abcam), GLS2 (Bio-Techne), phosphorylated mTOR (Serine 2448, p-mTORS2448 ; Cell Signaling Technologies), mTOR (Cell Signaling Technologies), phosphorylated p70 S6 kinase (Threonine 389, p-p70 S6KT389 ; Serine 371, p-p70 S6KS371 ; Cell Signaling Technologies), p70 S6K (Cell Signaling Technologies), phosphorylated S6 (Serine 235 and 236, S6S235/236 ; Cell Signaling Technologies), S6 (Cell Signaling Technologies), phosphorylated 4E-BP1 (Threonine 37/46, p-4E-BP1T37/46 ; Cell Signaling Technologies), 4E-BP1 (Cell Signaling Technologies), β-actin (Sigma-Aldrich) and Lamin A/C (Santa Cruz Biotechnologies).

    Techniques: Cell Culture, BrdU Incorporation Assay, Mouse Assay, Immunostaining, Derivative Assay

    Activation of PKM2 by TEPP46 attenuates mucus metaplasia, subepithelial collagen, and markers of airway remodeling in mice with HDM-induced allergic airway disease. ( A ) Assessment and quantification of mucus metaplasia by Periodic acid–Schiff (PAS) staining intensity and ( B ) collagen deposition by Masson’s trichrome staining. Scale bar, 200 μm. ( C ) mRNA expression of Muc5AC , and Col1a1 , normalized to Ppia . ( D ) Representative Western blots for SMA levels and the loading control β-actin. ( E ) Assessment of α-SMA staining around large airways ( n = 5 per group). Scale bar, 300 μm. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite–Induced Allergic Airways Disease

    doi: 10.4049/jimmunol.1901086

    Figure Lengend Snippet: Activation of PKM2 by TEPP46 attenuates mucus metaplasia, subepithelial collagen, and markers of airway remodeling in mice with HDM-induced allergic airway disease. ( A ) Assessment and quantification of mucus metaplasia by Periodic acid–Schiff (PAS) staining intensity and ( B ) collagen deposition by Masson’s trichrome staining. Scale bar, 200 μm. ( C ) mRNA expression of Muc5AC , and Col1a1 , normalized to Ppia . ( D ) Representative Western blots for SMA levels and the loading control β-actin. ( E ) Assessment of α-SMA staining around large airways ( n = 5 per group). Scale bar, 300 μm. * p

    Article Snippet: PKM1 (no. 7067), PKM2 (no. 4053), p-STAT3 (no. 8119), STAT3 (no. 4904), inhibitory κ B kinase ε (IKKε; no. 3416), and histone H3 (no. 4499) Abs were obtained from Cell Signaling Technology (Danvers, MA). β-Actin Ab was acquired from Sigma-Aldrich.

    Techniques: Activation Assay, Mouse Assay, Staining, Expressing, Western Blot

    TEPP46 augments PKM activity and PKM2’s cytosolic presence and attenuates IL-1β–mediated lactate secretion in primary MTE cells. MTE cells were treated with 100 μM TEPP46 for 1 h prior to stimulation with 10 ng/ml IL-1β for 24 h. ( A ) Representative Western blot of total PKM1 and PKM2 levels and β-actin. ( B ) PKM activity assay in MTE cells and ( C ) representative Western blot for tetrameric, dimeric, and monomeric PKM2 and the loading control β-actin. MTE cells were incubated in the presence or absence (first lane of each condition) of the disuccinimidyl suberate (DSS) cross-linker to evaluate the formation of the isoforms of PKM2. ( D ) Representative Western blots of nuclear and cytosolic extracts of PKM2 ( n = 3 per group). ( E ) Lactate levels in supernatants of MTE cells. Experiments were performed at least three times. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite–Induced Allergic Airways Disease

    doi: 10.4049/jimmunol.1901086

    Figure Lengend Snippet: TEPP46 augments PKM activity and PKM2’s cytosolic presence and attenuates IL-1β–mediated lactate secretion in primary MTE cells. MTE cells were treated with 100 μM TEPP46 for 1 h prior to stimulation with 10 ng/ml IL-1β for 24 h. ( A ) Representative Western blot of total PKM1 and PKM2 levels and β-actin. ( B ) PKM activity assay in MTE cells and ( C ) representative Western blot for tetrameric, dimeric, and monomeric PKM2 and the loading control β-actin. MTE cells were incubated in the presence or absence (first lane of each condition) of the disuccinimidyl suberate (DSS) cross-linker to evaluate the formation of the isoforms of PKM2. ( D ) Representative Western blots of nuclear and cytosolic extracts of PKM2 ( n = 3 per group). ( E ) Lactate levels in supernatants of MTE cells. Experiments were performed at least three times. * p

    Article Snippet: PKM1 (no. 7067), PKM2 (no. 4053), p-STAT3 (no. 8119), STAT3 (no. 4904), inhibitory κ B kinase ε (IKKε; no. 3416), and histone H3 (no. 4499) Abs were obtained from Cell Signaling Technology (Danvers, MA). β-Actin Ab was acquired from Sigma-Aldrich.

    Techniques: Activity Assay, Western Blot, Incubation

    Activation of PKM2 by TEPP46 attenuates proinflammatory cytokines in mice with HDM-induced allergic airway disease. ( A ) Schematic depicting the exposure regimen. Mice were sensitized twice with 10 μg of HDM or saline on days 1 and 8. Mice were treated with 50 mg/kg TEPP46 i.p. daily, starting on day 14. Mice were challenged with HDM on days 15–19 and euthanized 24 h after the final HDM challenge. ( B ) Assessment of PKM activity in lung tissue homogenates. ( C ) Representative Western blots and quantification for total PKM1 and PKM2 levels. β-Actin; loading control ( n = 3–6 per group). ( D and E ) Total and differential cell counts in BAL fluid. ( F ) Measurements of CCL20, IL-33, KC, and IL-1β in lung tissue homogenates by ELISA. (A, B, and D–F) n = 8–10 per group. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite–Induced Allergic Airways Disease

    doi: 10.4049/jimmunol.1901086

    Figure Lengend Snippet: Activation of PKM2 by TEPP46 attenuates proinflammatory cytokines in mice with HDM-induced allergic airway disease. ( A ) Schematic depicting the exposure regimen. Mice were sensitized twice with 10 μg of HDM or saline on days 1 and 8. Mice were treated with 50 mg/kg TEPP46 i.p. daily, starting on day 14. Mice were challenged with HDM on days 15–19 and euthanized 24 h after the final HDM challenge. ( B ) Assessment of PKM activity in lung tissue homogenates. ( C ) Representative Western blots and quantification for total PKM1 and PKM2 levels. β-Actin; loading control ( n = 3–6 per group). ( D and E ) Total and differential cell counts in BAL fluid. ( F ) Measurements of CCL20, IL-33, KC, and IL-1β in lung tissue homogenates by ELISA. (A, B, and D–F) n = 8–10 per group. * p

    Article Snippet: PKM1 (no. 7067), PKM2 (no. 4053), p-STAT3 (no. 8119), STAT3 (no. 4904), inhibitory κ B kinase ε (IKKε; no. 3416), and histone H3 (no. 4499) Abs were obtained from Cell Signaling Technology (Danvers, MA). β-Actin Ab was acquired from Sigma-Aldrich.

    Techniques: Activation Assay, Mouse Assay, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

    Effects of genetic deletion of p38 alpha and of SB203580 treatment on TcsL-catalyzed Rac/Cdc42 glucosylation (TcsL concentration-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with the indicated concentrations of TcsL in the presence of SB203580 (10 µM) or DMSO alone for 4 h. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, PAK2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three independent experiments. * indicates significant differences, p

    Journal: Toxins

    Article Title: Role of p38alpha/beta MAP Kinase in Cell Susceptibility to Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    doi: 10.3390/toxins9010002

    Figure Lengend Snippet: Effects of genetic deletion of p38 alpha and of SB203580 treatment on TcsL-catalyzed Rac/Cdc42 glucosylation (TcsL concentration-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with the indicated concentrations of TcsL in the presence of SB203580 (10 µM) or DMSO alone for 4 h. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, PAK2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three independent experiments. * indicates significant differences, p

    Article Snippet: Immunoblot Analysis Cells lysates were separated on 15% polyacrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA, followed by blocking with 5% (w /v ) nonfat dried milk for 1 h. Blots were incubated with the appropriate primary antibody with dilution according to the manufacturers’ instructions (beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5000); MAPKAPK-2 (Cell signaling 3042, dilution 1:1000); pT222-MAPKAPK-2 (Cell signaling 3316, dilution 1:1000); PAK2 (Cell signaling 2608, dilution 1:1000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:2000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1000); Rac1(Millipore 05-389, clone 23A8; dilution 1:1000) in buffer B (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w /v ) Tween 20) for 18 h and subsequently for 2 h with a horseradish peroxidase-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:3000; rabbit Rockland 611-1302; dilution 1:3000).

    Techniques: Concentration Assay, Plasmid Preparation, Western Blot, Software

    Effects of genetic deletion of p38 alpha on TcdB-catalyzed Rac/Cdc42 glucosylation (time-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with TcdB (1 ng/mL) for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three experiments.

    Journal: Toxins

    Article Title: Role of p38alpha/beta MAP Kinase in Cell Susceptibility to Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    doi: 10.3390/toxins9010002

    Figure Lengend Snippet: Effects of genetic deletion of p38 alpha on TcdB-catalyzed Rac/Cdc42 glucosylation (time-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with TcdB (1 ng/mL) for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three experiments.

    Article Snippet: Immunoblot Analysis Cells lysates were separated on 15% polyacrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA, followed by blocking with 5% (w /v ) nonfat dried milk for 1 h. Blots were incubated with the appropriate primary antibody with dilution according to the manufacturers’ instructions (beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5000); MAPKAPK-2 (Cell signaling 3042, dilution 1:1000); pT222-MAPKAPK-2 (Cell signaling 3316, dilution 1:1000); PAK2 (Cell signaling 2608, dilution 1:1000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:2000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1000); Rac1(Millipore 05-389, clone 23A8; dilution 1:1000) in buffer B (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w /v ) Tween 20) for 18 h and subsequently for 2 h with a horseradish peroxidase-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:3000; rabbit Rockland 611-1302; dilution 1:3000).

    Techniques: Plasmid Preparation, Western Blot, Software

    Effects of genetic deletion of p38 alpha and of SB203580 treatment on TcsL-catalyzed Rac/Cdc42 glucosylation (time-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with TcsL (1 µg/mL) in the presence of SB203580 (10 µM) or DMSO alone for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, PAK2, pT222-MAPKAPK2, MAPKAPK2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three independent experiments. * indicates significant differences, p

    Journal: Toxins

    Article Title: Role of p38alpha/beta MAP Kinase in Cell Susceptibility to Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    doi: 10.3390/toxins9010002

    Figure Lengend Snippet: Effects of genetic deletion of p38 alpha and of SB203580 treatment on TcsL-catalyzed Rac/Cdc42 glucosylation (time-dependency). p38 alpha −/− MSCV p38 alpha MEFs and p38 alpha −/− MSCV empty vector (EV) MEFs were treated with TcsL (1 µg/mL) in the presence of SB203580 (10 µM) or DMSO alone for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, PAK2, pT222-MAPKAPK2, MAPKAPK2, and beta-actin were analyzed by immunoblotting using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three independent experiments. * indicates significant differences, p

    Article Snippet: Immunoblot Analysis Cells lysates were separated on 15% polyacrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA, followed by blocking with 5% (w /v ) nonfat dried milk for 1 h. Blots were incubated with the appropriate primary antibody with dilution according to the manufacturers’ instructions (beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5000); MAPKAPK-2 (Cell signaling 3042, dilution 1:1000); pT222-MAPKAPK-2 (Cell signaling 3316, dilution 1:1000); PAK2 (Cell signaling 2608, dilution 1:1000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:2000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1000); Rac1(Millipore 05-389, clone 23A8; dilution 1:1000) in buffer B (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w /v ) Tween 20) for 18 h and subsequently for 2 h with a horseradish peroxidase-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:3000; rabbit Rockland 611-1302; dilution 1:3000).

    Techniques: Plasmid Preparation, Western Blot, Software

    Znhit1 deletion abrogates H2A/H2A.Z exchange to disrupt skewed lymphoid lineage commitment. a B6;129- Gt(ROSA)26Sor tm1(CAG-cas9*,−EGFP)Fezh /J knockin mice were crossed with Vav-Cre mice to obtain tissue-specific Cas9 expression. HSCs were sorted and infected with sg Znhit1 -containing lentivirus and then transplanted into lethally irradiated recipient mice (CD45.1) for 8 weeks. ZNHIT1 was detected by immunoblotting. β-Actin were used as a loading control. b Indicated MPPs were sorted and lysed for ChIP assays with anti-H2A.Z antibody. Indicated promoters were examined by real-time qPCR. Signals were normalized to input DNA. c Indicated LMPPs were sorted and co-cultured with OP9-DL1 stromal cells as in Fig. 2c . Data are representative of six independent transfection and transplantation mice for each group. ** P

    Journal: Nature Communications

    Article Title: Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

    doi: 10.1038/s41467-017-01788-7

    Figure Lengend Snippet: Znhit1 deletion abrogates H2A/H2A.Z exchange to disrupt skewed lymphoid lineage commitment. a B6;129- Gt(ROSA)26Sor tm1(CAG-cas9*,−EGFP)Fezh /J knockin mice were crossed with Vav-Cre mice to obtain tissue-specific Cas9 expression. HSCs were sorted and infected with sg Znhit1 -containing lentivirus and then transplanted into lethally irradiated recipient mice (CD45.1) for 8 weeks. ZNHIT1 was detected by immunoblotting. β-Actin were used as a loading control. b Indicated MPPs were sorted and lysed for ChIP assays with anti-H2A.Z antibody. Indicated promoters were examined by real-time qPCR. Signals were normalized to input DNA. c Indicated LMPPs were sorted and co-cultured with OP9-DL1 stromal cells as in Fig. 2c . Data are representative of six independent transfection and transplantation mice for each group. ** P

    Article Snippet: Antibodies against β-actin (AC-74) and His-tag (HIS-1) were from Sigma-Aldrich (St. Louis, USA).

    Techniques: Knock-In, Mouse Assay, Expressing, Infection, Irradiation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Transplantation Assay

    Mutations in the conserved regions of Jhd2 can affect its activity and overall protein levels. A , shown are a schematic representation and the domain locations of Jhd2. The three conserved domains, JmjN, PHD finger, and JmjC, are shown. Dark gray circles , residues that bind to the cofactor ferrous ion (Fe 2+ ); white circles , residues that bind to the cofactor α-ketoglutarate; light gray and black circles , a conserved residue (His 261 ) in the PHD finger and a conserved residue (Thr 359 ) corresponding to a human mental retardation-linked mutation site, respectively. Alignment of the Jhd2 sequence flanking Thr 359 and the SMCX sequence flanking one of the sites mutated in X-linked mental retardation (Ser 451 ) is also shown. B , shown are the results from Western blot analysis of H3K4 methylation levels in crude nuclear extracts from cells overexpressing WT JHD2 or its mutants. exp. , exposure. C , shown are the results from Western blot analysis of the levels of Jhd2–9Myc or its mutant derivatives in WCEs. The level of Pgk1 served as a control for total protein loading. D , WCEs were prepared from HeLa cells expressing SMCX -6Myc or the mutant ( smcx(S451R) -6Myc), and protein levels were examined using anti-Myc antibody. The level of β-actin served as a control for total protein loading.

    Journal: The Journal of Biological Chemistry

    Article Title: The JmjN Domain of Jhd2 Is Important for Its Protein Stability, and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation *

    doi: 10.1074/jbc.M110.117333

    Figure Lengend Snippet: Mutations in the conserved regions of Jhd2 can affect its activity and overall protein levels. A , shown are a schematic representation and the domain locations of Jhd2. The three conserved domains, JmjN, PHD finger, and JmjC, are shown. Dark gray circles , residues that bind to the cofactor ferrous ion (Fe 2+ ); white circles , residues that bind to the cofactor α-ketoglutarate; light gray and black circles , a conserved residue (His 261 ) in the PHD finger and a conserved residue (Thr 359 ) corresponding to a human mental retardation-linked mutation site, respectively. Alignment of the Jhd2 sequence flanking Thr 359 and the SMCX sequence flanking one of the sites mutated in X-linked mental retardation (Ser 451 ) is also shown. B , shown are the results from Western blot analysis of H3K4 methylation levels in crude nuclear extracts from cells overexpressing WT JHD2 or its mutants. exp. , exposure. C , shown are the results from Western blot analysis of the levels of Jhd2–9Myc or its mutant derivatives in WCEs. The level of Pgk1 served as a control for total protein loading. D , WCEs were prepared from HeLa cells expressing SMCX -6Myc or the mutant ( smcx(S451R) -6Myc), and protein levels were examined using anti-Myc antibody. The level of β-actin served as a control for total protein loading.

    Article Snippet: Following a 2-day incubation at 37 °C, cells were washed prior to and after harvesting with ice-cold 1× phosphate-buffered saline (Sigma) and boiled in 1× Laemmli sample buffer (Bio-Rad) for 10 min. After centrifugation at 16,100 × g for 5 min, equal volumes of WCEs were subjected to Western blot analysis using anti-Myc (1:1000) and anti-β-actin (1:10,000; Sigma) antibodies.

    Techniques: Activity Assay, Mutagenesis, Sequencing, Western Blot, Methylation, Expressing

    S100A10 is down-regulated by infection of Lv-miR-590-5P. ( A ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P. After 96 h, the cells were collected and total RNA was extracted and subjected to quantitative PCR. U6 was used as an internal standard; ( B ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P, and after 96 hours, the cells were collected and total protein was extracted and subjected to western blotting. For western blotting, cell lysates were separated on a 14% (S100A10) or 13% (beta-actin) SDS polyacrylamidegel, and the blots were detected with a specific anti-S100A10 antibody, with beta-actin as an internal standard **, t -test significant at p

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-590-5P Inhibits Growth of HepG2 Cells via Decrease of S100A10 Expression and Inhibition of the Wnt Pathway

    doi: 10.3390/ijms14048556

    Figure Lengend Snippet: S100A10 is down-regulated by infection of Lv-miR-590-5P. ( A ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P. After 96 h, the cells were collected and total RNA was extracted and subjected to quantitative PCR. U6 was used as an internal standard; ( B ) HepG2 cells were infected with Lv-control or Lv-miR-590-5P, and after 96 hours, the cells were collected and total protein was extracted and subjected to western blotting. For western blotting, cell lysates were separated on a 14% (S100A10) or 13% (beta-actin) SDS polyacrylamidegel, and the blots were detected with a specific anti-S100A10 antibody, with beta-actin as an internal standard **, t -test significant at p

    Article Snippet: The membranes were blocked with TBST containing 5% non-fat milk and incubated with antibodies against human S100A10 (1:200) (Santacruz) and beta-actin (1:1000) (Cell Signaling Technology) at 4 °C overnight.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Western Blot

    Thyroid hormone mediated ERK activation stimulates PIMT phosphorylation and enhances hepatic gluconeogenesis in rats. Rats (n=5) were injected intraperitoneally (0.1mL/animal) with L-thyroxine solution for 2 weeks. Post treatment animals were sacrificed, serum was collected for detecting levels of T 3 ( A ) and T 4 ( B ) and whole body weight ( C ) were measured. ( D ) PIMT immunoprecipitated lysates from liver tissue were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. ( E )PIMT was immunoprecipitated from liver tissue lysates (Set1: 3 control and 3 treated; Set2: 2 control and 2 treated) separated on 10% SDS PAGE and probed with Anti-MPM2 followed by Anti-PIMT as mentioned. ( F ) Chromatin immunoprecipitation assay was performed in liver tissue lysates (one control and one treated in both Set1 and Set2) using Anti-PIMT or mock Anti-goat IgG on rat PEPCK promoter. ( G ) The liver homogenates were analyzed using Anti-PEPCK (top panel) or Anti beta actin (bottom panel). ( H ) Overnight fasting blood sugar was measured using glucose strip.

    Journal: PLoS ONE

    Article Title: ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    doi: 10.1371/journal.pone.0083787

    Figure Lengend Snippet: Thyroid hormone mediated ERK activation stimulates PIMT phosphorylation and enhances hepatic gluconeogenesis in rats. Rats (n=5) were injected intraperitoneally (0.1mL/animal) with L-thyroxine solution for 2 weeks. Post treatment animals were sacrificed, serum was collected for detecting levels of T 3 ( A ) and T 4 ( B ) and whole body weight ( C ) were measured. ( D ) PIMT immunoprecipitated lysates from liver tissue were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. ( E )PIMT was immunoprecipitated from liver tissue lysates (Set1: 3 control and 3 treated; Set2: 2 control and 2 treated) separated on 10% SDS PAGE and probed with Anti-MPM2 followed by Anti-PIMT as mentioned. ( F ) Chromatin immunoprecipitation assay was performed in liver tissue lysates (one control and one treated in both Set1 and Set2) using Anti-PIMT or mock Anti-goat IgG on rat PEPCK promoter. ( G ) The liver homogenates were analyzed using Anti-PEPCK (top panel) or Anti beta actin (bottom panel). ( H ) Overnight fasting blood sugar was measured using glucose strip.

    Article Snippet: Western analysis was performed with Anti-GFP, Anti-pERK (1:1000), total ERK antibodies (1:1000), Anti- PEPCK (Santacruz Biotechnology Inc., Santacruz, CA, USA) and Anti-Beta actin (Cell Signaling Technology, Cambridge, MA, USA) and detected by ECL reagent (Amersham Biosciences, Piscataway, NJ, USA).

    Techniques: Activation Assay, Injection, Immunoprecipitation, Western Blot, SDS Page, Chromatin Immunoprecipitation, Stripping Membranes

    The expression of alpha-actinin-3 in human skeletal muscle and pulmonary artery smooth muscle. Shown are A) A rabbit polyclonal alpha-actinin 3 antibody probed against skeletal muscle samples from ACTN3 RR577 and 577XX individuals from the STRRIDE Study [ 30 ]. B ) This same antibody probed against human smooth muscle (SM) cell panel. Each lane contains 50 ug of protein extract, which were stripped and reprobed with a beta-actin antibody as a loading control.

    Journal: PLoS ONE

    Article Title: The ACTN3 R577X Polymorphism Is Associated with Cardiometabolic Fitness in Healthy Young Adults

    doi: 10.1371/journal.pone.0130644

    Figure Lengend Snippet: The expression of alpha-actinin-3 in human skeletal muscle and pulmonary artery smooth muscle. Shown are A) A rabbit polyclonal alpha-actinin 3 antibody probed against skeletal muscle samples from ACTN3 RR577 and 577XX individuals from the STRRIDE Study [ 30 ]. B ) This same antibody probed against human smooth muscle (SM) cell panel. Each lane contains 50 ug of protein extract, which were stripped and reprobed with a beta-actin antibody as a loading control.

    Article Snippet: All blots were subsequently stripped and re-probed with an beta-actin antibody (Cell Signaling) as a loading control as described previously [ ].To show the specificity of the α-actinin 3 primary antibody, previously genotyped human skeletal muscle samples (50 ug each) from the STRRIDE study [ ] were similarly probed with each antibody.

    Techniques: Expressing

    Expression of proteins which were reported to be associated with sensitivity to anti-microtubule agents. (A) Protein expression was evaluated by western blot analysis. Expression values of class Ⅲ beta-tubulin (TUBB3) relative to beta-actin were determined using Just TLC software. (B) Representative images of HCC4006 and HCC4006ER cells immunohistochemically stained with antibodies to ATP-binding cassette subfamily B, member 1 (ABCB1).

    Journal: PLoS ONE

    Article Title: Collateral Chemoresistance to Anti-Microtubule Agents in a Lung Cancer Cell Line with Acquired Resistance to Erlotinib

    doi: 10.1371/journal.pone.0123901

    Figure Lengend Snippet: Expression of proteins which were reported to be associated with sensitivity to anti-microtubule agents. (A) Protein expression was evaluated by western blot analysis. Expression values of class Ⅲ beta-tubulin (TUBB3) relative to beta-actin were determined using Just TLC software. (B) Representative images of HCC4006 and HCC4006ER cells immunohistochemically stained with antibodies to ATP-binding cassette subfamily B, member 1 (ABCB1).

    Article Snippet: Antibodies and western blot analysis Anti-E-cadherin, anti-ATP-binding cassette subfamily B, member 1 (ABCB1), anti-class III beta-tubulin (TUBB3) and anti-beta-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, Western Blot, Thin Layer Chromatography, Software, Staining, Binding Assay

    Western blot of DNMT1 expression. This image is a western blot of DNMT1 protein expression in CLGL 90 and CLL 17–7 cells treated with 1.5 μM or 6 μM 6-TG or 50 μM or 200 μM Zeb concentrations when compared to control. DNMT bands are evident at approximately 180,000 molecular weight. The treated groups had a reduction in intensity of DNMT1 expression as evident by bands in the 180 kDA region. Reduction in the expression of DNMT1 treated CLGL 90 cells (6-TG low, high and Zeb low, high groups) was 0.81, 0.76, 0.89, and 0.67 when compared to untreated control, respectively. Reduction in the expression of DNMT1 treated CLL 17–7 cells (6-TG and Zeb high dose) is 0.27 and 0.40, respectively. Loading control is Beta-actin.

    Journal: BMC Veterinary Research

    Article Title: 6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells

    doi: 10.1186/s12917-014-0290-8

    Figure Lengend Snippet: Western blot of DNMT1 expression. This image is a western blot of DNMT1 protein expression in CLGL 90 and CLL 17–7 cells treated with 1.5 μM or 6 μM 6-TG or 50 μM or 200 μM Zeb concentrations when compared to control. DNMT bands are evident at approximately 180,000 molecular weight. The treated groups had a reduction in intensity of DNMT1 expression as evident by bands in the 180 kDA region. Reduction in the expression of DNMT1 treated CLGL 90 cells (6-TG low, high and Zeb low, high groups) was 0.81, 0.76, 0.89, and 0.67 when compared to untreated control, respectively. Reduction in the expression of DNMT1 treated CLL 17–7 cells (6-TG and Zeb high dose) is 0.27 and 0.40, respectively. Loading control is Beta-actin.

    Article Snippet: The blots were re-probed with antibody against Beta-actin (#4970; 21 μg/ml; 1:1000) (Cell Signaling Technologies), which served as a loading control.

    Techniques: Western Blot, Expressing, Molecular Weight

    NT-Sr promote RANKL-induced phosphorylation of ERK and inhibit the Akt/NFATc1 pathway. RAW264.7 cells were cultured on different samples for 3 d and stimulated with or without 100 ng/mL RANKL for 30 min, and the total protein was then collected for immunoblot analysis. ( a ) RANKL-induced phosphorylation of ERK, ( b ) p-Akt and NFATc1 were detected using β-actin, total ERK and Akt as loading controls. ( c ) A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. Full-length blots are presented in Supplementary Figure 4 . The numbers 1, 2, 3 and 4 in the figure represent Ti, TiO 2 -NTs, NT-Sr1h and NT-Sr3h, respectively. * , **p

    Journal: Scientific Reports

    Article Title: Strontium-loaded titania nanotube arrays repress osteoclast differentiation through multiple signalling pathways: In vitro and in vivo studies

    doi: 10.1038/s41598-017-02491-9

    Figure Lengend Snippet: NT-Sr promote RANKL-induced phosphorylation of ERK and inhibit the Akt/NFATc1 pathway. RAW264.7 cells were cultured on different samples for 3 d and stimulated with or without 100 ng/mL RANKL for 30 min, and the total protein was then collected for immunoblot analysis. ( a ) RANKL-induced phosphorylation of ERK, ( b ) p-Akt and NFATc1 were detected using β-actin, total ERK and Akt as loading controls. ( c ) A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. Full-length blots are presented in Supplementary Figure 4 . The numbers 1, 2, 3 and 4 in the figure represent Ti, TiO 2 -NTs, NT-Sr1h and NT-Sr3h, respectively. * , **p

    Article Snippet: The antibody against β-actin (β-actin) and secondary antibodies were purchased from BOSTER (Wuhan, China, 1:5000 dilution).

    Techniques: Cell Culture

    NT-Sr repress osteoclast-specific genes. RAW264.7 cells ( a ) and mouse BMMCs ( b ) were cultured on different samples and induced with 50 ng/mL RANKL and 30 ng/mL M-CSF (for BMMCs), and the total RNA was then collected. The relative mRNA expression levels of osteoclast-specific genes (TRAP, CK, MMP-9, and NFATc1) were assessed by qRT-PCR. For total protein extraction, RAW264.7 cells ( c , e ) and mouse BMMCs ( d , f ) were cultured on different samples in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml, for BMMCs). The protein expression of osteoclast markers (TRAP, CK, and MMP-9) was then detected by immunoblotting. An antibody to β-actin was used as a loading control. A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading control. Full-length blots are presented in Supplementary Figure 2 . Sr1h and Sr3h represent NT-Sr1h and NT-Sr3h, respectively. * , **p

    Journal: Scientific Reports

    Article Title: Strontium-loaded titania nanotube arrays repress osteoclast differentiation through multiple signalling pathways: In vitro and in vivo studies

    doi: 10.1038/s41598-017-02491-9

    Figure Lengend Snippet: NT-Sr repress osteoclast-specific genes. RAW264.7 cells ( a ) and mouse BMMCs ( b ) were cultured on different samples and induced with 50 ng/mL RANKL and 30 ng/mL M-CSF (for BMMCs), and the total RNA was then collected. The relative mRNA expression levels of osteoclast-specific genes (TRAP, CK, MMP-9, and NFATc1) were assessed by qRT-PCR. For total protein extraction, RAW264.7 cells ( c , e ) and mouse BMMCs ( d , f ) were cultured on different samples in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml, for BMMCs). The protein expression of osteoclast markers (TRAP, CK, and MMP-9) was then detected by immunoblotting. An antibody to β-actin was used as a loading control. A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading control. Full-length blots are presented in Supplementary Figure 2 . Sr1h and Sr3h represent NT-Sr1h and NT-Sr3h, respectively. * , **p

    Article Snippet: The antibody against β-actin (β-actin) and secondary antibodies were purchased from BOSTER (Wuhan, China, 1:5000 dilution).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Protein Extraction

    NT-Sr inhibit RANKL-induced NF-κB activation. After RAW264.7 cells ( a , b ) and mouse BMMCs ( e , f ) were cultured on different samples for 3 d, the cells were stimulated with or without 100 ng/mL RANKL for 30 min, and the total proteins were extracted for western blot analysis. The expression of proteins in the NF-κB pathway and the levels of p-IKKβ, p-IκBα, and p-NF-κBp65 were detected. Antibodies against β-actin, total IKKβ, IκBα, and NF-κBp65 were used as loading controls. A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. RAW264.7 cells ( c , d ) were cultured on different samples for 3 d and stimulated with 100 ng/mL RANKL for 30 min. The nuclear extracts were then collected, and the DNA-binding activity of NF-κB was detected by electrophoretic mobility shift assay (EMSA). A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. Full-length blots are presented in Supplementary Figure 3 . p-p65 and p65 represent p-NF-κBp65 and NF-κBp65, respectively; and the numbers 1, 2, 3 and 4 in the figure represent Ti, TiO 2 -NTs, NT-Sr1h and NT-Sr3h, respectively. * , **p

    Journal: Scientific Reports

    Article Title: Strontium-loaded titania nanotube arrays repress osteoclast differentiation through multiple signalling pathways: In vitro and in vivo studies

    doi: 10.1038/s41598-017-02491-9

    Figure Lengend Snippet: NT-Sr inhibit RANKL-induced NF-κB activation. After RAW264.7 cells ( a , b ) and mouse BMMCs ( e , f ) were cultured on different samples for 3 d, the cells were stimulated with or without 100 ng/mL RANKL for 30 min, and the total proteins were extracted for western blot analysis. The expression of proteins in the NF-κB pathway and the levels of p-IKKβ, p-IκBα, and p-NF-κBp65 were detected. Antibodies against β-actin, total IKKβ, IκBα, and NF-κBp65 were used as loading controls. A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. RAW264.7 cells ( c , d ) were cultured on different samples for 3 d and stimulated with 100 ng/mL RANKL for 30 min. The nuclear extracts were then collected, and the DNA-binding activity of NF-κB was detected by electrophoretic mobility shift assay (EMSA). A quantitative analysis of the band densities was performed, and the band densities were normalised to the loading controls. Full-length blots are presented in Supplementary Figure 3 . p-p65 and p65 represent p-NF-κBp65 and NF-κBp65, respectively; and the numbers 1, 2, 3 and 4 in the figure represent Ti, TiO 2 -NTs, NT-Sr1h and NT-Sr3h, respectively. * , **p

    Article Snippet: The antibody against β-actin (β-actin) and secondary antibodies were purchased from BOSTER (Wuhan, China, 1:5000 dilution).

    Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay

    TDD‐mediated regulation of HMBOX1 expression in EA.hy926 cells. (A) HMBOX1 mRNA expression was analyzed by FQ‐PCR and normalized mRNA values relative to GAPDH levels were plotted. (B) Effect of TDD on HMBOX1 protein levels was evaluated by western blot with analysis of β‐actin expression as loading control. The data show the mean ± S.D. of three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction, et al. 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

    doi: 10.1111/jcmm.13948

    Figure Lengend Snippet: TDD‐mediated regulation of HMBOX1 expression in EA.hy926 cells. (A) HMBOX1 mRNA expression was analyzed by FQ‐PCR and normalized mRNA values relative to GAPDH levels were plotted. (B) Effect of TDD on HMBOX1 protein levels was evaluated by western blot with analysis of β‐actin expression as loading control. The data show the mean ± S.D. of three independent experiments. * P

    Article Snippet: ELISA kits for IL‐6 and TNF‐α, β‐actin antibody, horseradish peroxidase‐conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection kit were obtained from Boster Biotech Co., Ltd. (Wuhan, China).

    Techniques: Expressing, Polymerase Chain Reaction, Western Blot

    Extracellular AGR2 enhances RhoA, CDC42 expression and stimulates FAK phosphorylation in NIH3T3 cells. (a) Western blot analysis of RhoA expression in NIH3T3 cells stimulated for 24 h with AGR2 coupled with or without bFGF. The concentration of AGR2 is 500 ng/ml. The concentration of bFGF is 1 ng/ml. (b) NIH3T3 cells were treated by 500 ng/ml AGR2 for the indicated time. RhoA, cyclin D1, P21, ERK1/2, pERK1/2, CDC42, Rac1, RhoB, and RhoC were detected by western blots. β-actin served as loading control. (c) Significant reduction in RhoA expression, cyclin D1 expression, and FAK phosphorylation was observed in the NIH3T3 cells pretreated with 20 nM/ml FGFR1 inhibitor PD173074 and 10 nM/ml VEGFR inhibitor axitinib before AGR2 treatment. β-actin served as loading control.

    Journal: Cell Adhesion & Migration

    Article Title: Paracrine signalling of AGR2 stimulates RhoA function in fibroblasts and modulates cell elongation and migration

    doi: 10.1080/19336918.2019.1685928

    Figure Lengend Snippet: Extracellular AGR2 enhances RhoA, CDC42 expression and stimulates FAK phosphorylation in NIH3T3 cells. (a) Western blot analysis of RhoA expression in NIH3T3 cells stimulated for 24 h with AGR2 coupled with or without bFGF. The concentration of AGR2 is 500 ng/ml. The concentration of bFGF is 1 ng/ml. (b) NIH3T3 cells were treated by 500 ng/ml AGR2 for the indicated time. RhoA, cyclin D1, P21, ERK1/2, pERK1/2, CDC42, Rac1, RhoB, and RhoC were detected by western blots. β-actin served as loading control. (c) Significant reduction in RhoA expression, cyclin D1 expression, and FAK phosphorylation was observed in the NIH3T3 cells pretreated with 20 nM/ml FGFR1 inhibitor PD173074 and 10 nM/ml VEGFR inhibitor axitinib before AGR2 treatment. β-actin served as loading control.

    Article Snippet: Immunoblotting was performed as described previously with RhoA, p-FAK, Cyclin D1, p-ERK, ERK, P21 primary antibody (Cell signalling technology Inc., Denver, MA, USA), FAK, RhoB, RhoC, Rac1 Primary antibody (ABclonal, Woburn, MA, USA), CDC42 primary antibody (Abcam, Cambridge, MA, USA) and β-actin purchased from Abgent [ ].

    Techniques: Expressing, Western Blot, Concentration Assay