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  • 99
    Zymo Research total rna
    Total Rna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 7667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNA 5 µg Cecum Adenoma of cecum
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    Thermo Fisher rna
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 181696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 471882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa total rna
    Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 46719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore total rna
    Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr total rna
    Quantitative Real Time Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 16065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen total rna
    Total Rna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 275349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna extraction
    Total Rna Extraction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total cellular rna
    Total Cellular Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15858 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr total rna
    ZIP14-mediated zinc accumulation blocks muscle-cell differentiation and induces myosin heavy chain loss (a) <t>qRT-PCR</t> analysis of Zip14 expression in purified muscle progenitor subpopulations from either non-tumor-bearing control mice (Con) or mice bearing 4T1 or C26m2 metastases (Tb) harvested five weeks after tumor-cell injection (see Fig. 1a and Supplementary Fig. 1c ). CD45 − CD31 − CD34 + Sca1 + and CD45 − CD31 − CD34 + integrin-α7 + cells were purified from gastrocnemius muscles using a combination of magnetic and flow-cytometry-assisted sorting (see Supplementary Fig. 5a ). For 4T1 model, n=4 control mice for isolated CD34 + Sca1 + cells and n=3 mice for the other groups. For C26m2 model, n=3 mice per group. (b) ZIP14 immunofluorescence analysis using muscle sections from either non-tumor-bearing control mice or mice bearing C26m2 metastases five weeks after tumor-cell injection. ZIP14 (red), DAPI (blue). Scale bars, 50 μm. Boxed area in upper panels is magnified in the corresponding lower panels. A representative image from three independent experiments is shown. (c,d) Immunofluorescence analysis showing myosin heavy chain (MyHC) expression in C2C12 myoblasts infected with adenovirus expressing either control (Adeno-Con) or Zip14 cDNA (Adeno- Zip14 ) and differentiated for 6 days, either with 0 or 50 μM ZnCl 2 (zinc) replenished daily. Representative images and quantitation (d) are shown. Scale bars, 25 μm. Data presented as percentage of MyHC fluorescence signal in corresponding untreated myotubes (d). Data representative of three independent experiments (c,d). (e) qRT-PCR analysis of MyoD , Myf5 and Mef2c expression in untreated and zinc-treated C2C12 cells expressing either Adeno-Con or Adeno- Zip14 , represented as a heatmap. Adenovirus-infected C2C12 cells were differentiated for 2 days and then treated with either 0 or 50 μM ZnCl 2 (zinc) for 24 hours. Data is representative of four independent experiments. (f) <t>RNA-Seq</t> analysis of MyoD , Myf5 and Mef2c expression shown as heatmap (RNA seq shown in Fig. 2 and Supplementary Table 1 ) comparing TA muscles from non-tumor-bearing control mice to mice bearing 4T1 or C26m2 metastases, collected 5 weeks post tumor-cell injection. (g) MyHC and tropomyosin (Tm) protein expression by immunoblot analysis in C2C12 cells infected with adenovirus expressing either control or Zip14 cDNA and differentiated for 3 days followed by treatment with either 0 or 50 μM ZnCl 2 for 24 hours. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (h) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from mice intramuscularly injected with adeno-associated virus expressing either shCon or sh Zip14 and subsequently injected with C26m2 cancer cells (see Fig. 4d ). Age-matched, non-tumor-bearing mice were used as a control. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (i) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from the indicated groups. Muscles were isolated from Zip14 WT and KO mice with (Tb) or without (Con) 4T1 metastases. Another cohort of Zip14 KO mice were injected intramuscularly with AAV-Con ( mCherry ) or AAV- Zip14 in the gastrocnemius muscle and injected four weeks later with 4T1 tumor cells. All mice were harvested 5 weeks post tumor-cell injection. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . Error bars represent SEM and all data were represented by mean ± SEM. P values in (a,d) were determined by two-tailed, unpaired Student’s t test. Con, control; Tb, tumor-bearing; Tm, Tropomyosin. (j) Working model: During cancer progression and metastasis development, cytokines such as TNF-α and TGF-β upregulate the expression of Zip14 , a metal ion transporter, in muscle progenitor and mature muscle cells. This causes an aberrant accumulation of zinc in these muscle cells. ZIP14 expression and zinc uptake in muscle progenitor cells represses key myogenic genes such as MyoD and Mef2c , and blocks muscle differentiation. ZIP14 expression in mature muscle cells causes myosin heavy chain loss, which promotes cancer-induced muscle atrophy in metastatic cancers. Tf, tumor factors; Ca, cancer cells; Nr, normal cells.
    Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 9991 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad total rna
    ZIP14-mediated zinc accumulation blocks muscle-cell differentiation and induces myosin heavy chain loss (a) <t>qRT-PCR</t> analysis of Zip14 expression in purified muscle progenitor subpopulations from either non-tumor-bearing control mice (Con) or mice bearing 4T1 or C26m2 metastases (Tb) harvested five weeks after tumor-cell injection (see Fig. 1a and Supplementary Fig. 1c ). CD45 − CD31 − CD34 + Sca1 + and CD45 − CD31 − CD34 + integrin-α7 + cells were purified from gastrocnemius muscles using a combination of magnetic and flow-cytometry-assisted sorting (see Supplementary Fig. 5a ). For 4T1 model, n=4 control mice for isolated CD34 + Sca1 + cells and n=3 mice for the other groups. For C26m2 model, n=3 mice per group. (b) ZIP14 immunofluorescence analysis using muscle sections from either non-tumor-bearing control mice or mice bearing C26m2 metastases five weeks after tumor-cell injection. ZIP14 (red), DAPI (blue). Scale bars, 50 μm. Boxed area in upper panels is magnified in the corresponding lower panels. A representative image from three independent experiments is shown. (c,d) Immunofluorescence analysis showing myosin heavy chain (MyHC) expression in C2C12 myoblasts infected with adenovirus expressing either control (Adeno-Con) or Zip14 cDNA (Adeno- Zip14 ) and differentiated for 6 days, either with 0 or 50 μM ZnCl 2 (zinc) replenished daily. Representative images and quantitation (d) are shown. Scale bars, 25 μm. Data presented as percentage of MyHC fluorescence signal in corresponding untreated myotubes (d). Data representative of three independent experiments (c,d). (e) qRT-PCR analysis of MyoD , Myf5 and Mef2c expression in untreated and zinc-treated C2C12 cells expressing either Adeno-Con or Adeno- Zip14 , represented as a heatmap. Adenovirus-infected C2C12 cells were differentiated for 2 days and then treated with either 0 or 50 μM ZnCl 2 (zinc) for 24 hours. Data is representative of four independent experiments. (f) <t>RNA-Seq</t> analysis of MyoD , Myf5 and Mef2c expression shown as heatmap (RNA seq shown in Fig. 2 and Supplementary Table 1 ) comparing TA muscles from non-tumor-bearing control mice to mice bearing 4T1 or C26m2 metastases, collected 5 weeks post tumor-cell injection. (g) MyHC and tropomyosin (Tm) protein expression by immunoblot analysis in C2C12 cells infected with adenovirus expressing either control or Zip14 cDNA and differentiated for 3 days followed by treatment with either 0 or 50 μM ZnCl 2 for 24 hours. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (h) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from mice intramuscularly injected with adeno-associated virus expressing either shCon or sh Zip14 and subsequently injected with C26m2 cancer cells (see Fig. 4d ). Age-matched, non-tumor-bearing mice were used as a control. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (i) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from the indicated groups. Muscles were isolated from Zip14 WT and KO mice with (Tb) or without (Con) 4T1 metastases. Another cohort of Zip14 KO mice were injected intramuscularly with AAV-Con ( mCherry ) or AAV- Zip14 in the gastrocnemius muscle and injected four weeks later with 4T1 tumor cells. All mice were harvested 5 weeks post tumor-cell injection. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . Error bars represent SEM and all data were represented by mean ± SEM. P values in (a,d) were determined by two-tailed, unpaired Student’s t test. Con, control; Tb, tumor-bearing; Tm, Tropomyosin. (j) Working model: During cancer progression and metastasis development, cytokines such as TNF-α and TGF-β upregulate the expression of Zip14 , a metal ion transporter, in muscle progenitor and mature muscle cells. This causes an aberrant accumulation of zinc in these muscle cells. ZIP14 expression and zinc uptake in muscle progenitor cells represses key myogenic genes such as MyoD and Mef2c , and blocks muscle differentiation. ZIP14 expression in mature muscle cells causes myosin heavy chain loss, which promotes cancer-induced muscle atrophy in metastatic cancers. Tf, tumor factors; Ca, cancer cells; Nr, normal cells.
    Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 17279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna isolation
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Total Rna Isolation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 5996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore spectrum plant total rna kit
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Spectrum Plant Total Rna Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous kit
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Rnaqueous Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous micro kit
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Rnaqueous Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5931 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna synthesis total rna
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Cdna Synthesis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 4336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mrna
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 89710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc truseq stranded mrna library prep kit
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Truseq Stranded Mrna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna extraction total rna
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
    Rna Extraction Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 3291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute mammalian total rna miniprep kit
    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for <t>RNA</t> isolation and <t>qRT-PCR</t> analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P
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    ZIP14-mediated zinc accumulation blocks muscle-cell differentiation and induces myosin heavy chain loss (a) qRT-PCR analysis of Zip14 expression in purified muscle progenitor subpopulations from either non-tumor-bearing control mice (Con) or mice bearing 4T1 or C26m2 metastases (Tb) harvested five weeks after tumor-cell injection (see Fig. 1a and Supplementary Fig. 1c ). CD45 − CD31 − CD34 + Sca1 + and CD45 − CD31 − CD34 + integrin-α7 + cells were purified from gastrocnemius muscles using a combination of magnetic and flow-cytometry-assisted sorting (see Supplementary Fig. 5a ). For 4T1 model, n=4 control mice for isolated CD34 + Sca1 + cells and n=3 mice for the other groups. For C26m2 model, n=3 mice per group. (b) ZIP14 immunofluorescence analysis using muscle sections from either non-tumor-bearing control mice or mice bearing C26m2 metastases five weeks after tumor-cell injection. ZIP14 (red), DAPI (blue). Scale bars, 50 μm. Boxed area in upper panels is magnified in the corresponding lower panels. A representative image from three independent experiments is shown. (c,d) Immunofluorescence analysis showing myosin heavy chain (MyHC) expression in C2C12 myoblasts infected with adenovirus expressing either control (Adeno-Con) or Zip14 cDNA (Adeno- Zip14 ) and differentiated for 6 days, either with 0 or 50 μM ZnCl 2 (zinc) replenished daily. Representative images and quantitation (d) are shown. Scale bars, 25 μm. Data presented as percentage of MyHC fluorescence signal in corresponding untreated myotubes (d). Data representative of three independent experiments (c,d). (e) qRT-PCR analysis of MyoD , Myf5 and Mef2c expression in untreated and zinc-treated C2C12 cells expressing either Adeno-Con or Adeno- Zip14 , represented as a heatmap. Adenovirus-infected C2C12 cells were differentiated for 2 days and then treated with either 0 or 50 μM ZnCl 2 (zinc) for 24 hours. Data is representative of four independent experiments. (f) RNA-Seq analysis of MyoD , Myf5 and Mef2c expression shown as heatmap (RNA seq shown in Fig. 2 and Supplementary Table 1 ) comparing TA muscles from non-tumor-bearing control mice to mice bearing 4T1 or C26m2 metastases, collected 5 weeks post tumor-cell injection. (g) MyHC and tropomyosin (Tm) protein expression by immunoblot analysis in C2C12 cells infected with adenovirus expressing either control or Zip14 cDNA and differentiated for 3 days followed by treatment with either 0 or 50 μM ZnCl 2 for 24 hours. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (h) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from mice intramuscularly injected with adeno-associated virus expressing either shCon or sh Zip14 and subsequently injected with C26m2 cancer cells (see Fig. 4d ). Age-matched, non-tumor-bearing mice were used as a control. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (i) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from the indicated groups. Muscles were isolated from Zip14 WT and KO mice with (Tb) or without (Con) 4T1 metastases. Another cohort of Zip14 KO mice were injected intramuscularly with AAV-Con ( mCherry ) or AAV- Zip14 in the gastrocnemius muscle and injected four weeks later with 4T1 tumor cells. All mice were harvested 5 weeks post tumor-cell injection. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . Error bars represent SEM and all data were represented by mean ± SEM. P values in (a,d) were determined by two-tailed, unpaired Student’s t test. Con, control; Tb, tumor-bearing; Tm, Tropomyosin. (j) Working model: During cancer progression and metastasis development, cytokines such as TNF-α and TGF-β upregulate the expression of Zip14 , a metal ion transporter, in muscle progenitor and mature muscle cells. This causes an aberrant accumulation of zinc in these muscle cells. ZIP14 expression and zinc uptake in muscle progenitor cells represses key myogenic genes such as MyoD and Mef2c , and blocks muscle differentiation. ZIP14 expression in mature muscle cells causes myosin heavy chain loss, which promotes cancer-induced muscle atrophy in metastatic cancers. Tf, tumor factors; Ca, cancer cells; Nr, normal cells.

    Journal: Nature medicine

    Article Title: Metastatic cancers promote cachexia through altered zinc homeostasis in skeletal muscle

    doi: 10.1038/s41591-018-0054-2

    Figure Lengend Snippet: ZIP14-mediated zinc accumulation blocks muscle-cell differentiation and induces myosin heavy chain loss (a) qRT-PCR analysis of Zip14 expression in purified muscle progenitor subpopulations from either non-tumor-bearing control mice (Con) or mice bearing 4T1 or C26m2 metastases (Tb) harvested five weeks after tumor-cell injection (see Fig. 1a and Supplementary Fig. 1c ). CD45 − CD31 − CD34 + Sca1 + and CD45 − CD31 − CD34 + integrin-α7 + cells were purified from gastrocnemius muscles using a combination of magnetic and flow-cytometry-assisted sorting (see Supplementary Fig. 5a ). For 4T1 model, n=4 control mice for isolated CD34 + Sca1 + cells and n=3 mice for the other groups. For C26m2 model, n=3 mice per group. (b) ZIP14 immunofluorescence analysis using muscle sections from either non-tumor-bearing control mice or mice bearing C26m2 metastases five weeks after tumor-cell injection. ZIP14 (red), DAPI (blue). Scale bars, 50 μm. Boxed area in upper panels is magnified in the corresponding lower panels. A representative image from three independent experiments is shown. (c,d) Immunofluorescence analysis showing myosin heavy chain (MyHC) expression in C2C12 myoblasts infected with adenovirus expressing either control (Adeno-Con) or Zip14 cDNA (Adeno- Zip14 ) and differentiated for 6 days, either with 0 or 50 μM ZnCl 2 (zinc) replenished daily. Representative images and quantitation (d) are shown. Scale bars, 25 μm. Data presented as percentage of MyHC fluorescence signal in corresponding untreated myotubes (d). Data representative of three independent experiments (c,d). (e) qRT-PCR analysis of MyoD , Myf5 and Mef2c expression in untreated and zinc-treated C2C12 cells expressing either Adeno-Con or Adeno- Zip14 , represented as a heatmap. Adenovirus-infected C2C12 cells were differentiated for 2 days and then treated with either 0 or 50 μM ZnCl 2 (zinc) for 24 hours. Data is representative of four independent experiments. (f) RNA-Seq analysis of MyoD , Myf5 and Mef2c expression shown as heatmap (RNA seq shown in Fig. 2 and Supplementary Table 1 ) comparing TA muscles from non-tumor-bearing control mice to mice bearing 4T1 or C26m2 metastases, collected 5 weeks post tumor-cell injection. (g) MyHC and tropomyosin (Tm) protein expression by immunoblot analysis in C2C12 cells infected with adenovirus expressing either control or Zip14 cDNA and differentiated for 3 days followed by treatment with either 0 or 50 μM ZnCl 2 for 24 hours. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (h) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from mice intramuscularly injected with adeno-associated virus expressing either shCon or sh Zip14 and subsequently injected with C26m2 cancer cells (see Fig. 4d ). Age-matched, non-tumor-bearing mice were used as a control. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (i) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from the indicated groups. Muscles were isolated from Zip14 WT and KO mice with (Tb) or without (Con) 4T1 metastases. Another cohort of Zip14 KO mice were injected intramuscularly with AAV-Con ( mCherry ) or AAV- Zip14 in the gastrocnemius muscle and injected four weeks later with 4T1 tumor cells. All mice were harvested 5 weeks post tumor-cell injection. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . Error bars represent SEM and all data were represented by mean ± SEM. P values in (a,d) were determined by two-tailed, unpaired Student’s t test. Con, control; Tb, tumor-bearing; Tm, Tropomyosin. (j) Working model: During cancer progression and metastasis development, cytokines such as TNF-α and TGF-β upregulate the expression of Zip14 , a metal ion transporter, in muscle progenitor and mature muscle cells. This causes an aberrant accumulation of zinc in these muscle cells. ZIP14 expression and zinc uptake in muscle progenitor cells represses key myogenic genes such as MyoD and Mef2c , and blocks muscle differentiation. ZIP14 expression in mature muscle cells causes myosin heavy chain loss, which promotes cancer-induced muscle atrophy in metastatic cancers. Tf, tumor factors; Ca, cancer cells; Nr, normal cells.

    Article Snippet: RNA extraction for RNA sequencing and qRT-PCR Total RNA was extracted using Trizol® reagent (Thermo Fisher) as previously described .

    Techniques: Cell Differentiation, Quantitative RT-PCR, Expressing, Purification, Mouse Assay, Injection, Flow Cytometry, Cytometry, Isolation, Immunofluorescence, Infection, Quantitation Assay, Fluorescence, RNA Sequencing Assay, Two Tailed Test

    ZIP14 is upregulated in cachectic muscles from metastatic mouse models and patients, and is induced by TNF-α and TGF-β (a,b) Body weight measurements (a) and qRT-PCR analysis of Trim63 , MAFbx , Fbxo31 , Musa1 and Zip14 in TA muscles (b) derived from four independent metastatic lung cancer models, compared to respective age-matched controls. Metastatic models include conditional Kras/p53 mutant ( Kras LSL-G12D/+ - p53 fl/fl ) and Kras/Lkb1 mutant ( Kras LSL-G12D/+ - Lkb1 fl/fl ) in which muscles were collected at 13 weeks post adeno-Cre induction, PC9-BrM3 xenograft in which muscles were collected at 7 weeks post tumor-cell injection, and Rb/p53 mutant allografts in which muscles were collected at 6 weeks post tumor resection (see also Supplementary Fig. 3a ). For body weight analysis in (a), n=4 controls and n=8 Kras/p53 conditional mutant mice, n=3 controls and n=6 Kras/Lkb1 conditional mutant mice, n=3 controls and n=10 PC9-BrM3 xenograft mice, and n=3 controls and n=10 Rb/p53 mutant allograft mice. For qRT-PCR analysis in (b), n=6 controls and n=4 Kras/p53 conditional mutant mice, n=3 controls and n=4 Kras/Lkb1 conditional mutant mice, n=4 controls and n=5 PC9-BrM3 xenograft mice, and n=5 controls and n=4 Rb/p53 mutant allograft model mice. (c) Representative images of ZIP14 immunohistochemistry on human muscle cross-sections from non-cachectic (upper panel) and cachectic (lower panel) metastatic cancer patients. ZIP14 antibody details are shown in Supplementary Fig. 3f-h . Scale bars, 50 μm. ca, cancer. Patient details are listed in Supplementary Table 3 . Data is representative of three independent experiments. (d) qRT-PCR analysis of Zip14 in human skeletal primary muscle cells treated with either vehicle, TNF-α (50 ng/ml), TGF-β (10 ng/ml), or both TNF-α (50 ng/ml) + TGF-β (10 ng/ml), either alone (vehicle) or in the presence of 10 μM of the indicated inhibitors. Cells were pretreated with either vehicle or the indicated inhibitors for 1 hour prior to adding the cytokines (TGF-β for 9 hours, and TNF-α for 3 hours before harvest). Cells from all groups were harvested at the same time. n=6 samples for cells treated with vehicle, TGF-β alone or TNF-α together with TGFβ; n=4 samples for all the other groups. Inhibitors: NF-κBi = NF-κB inhibitor (BAY 11-7085); AP1i = AP1 inhibitor (CC401); Smadi = TGFβ receptor I inhibitor (SB431542). (e,f) qRT-PCR analysis of Zip14 in TA muscles after neutralizing antibody treatment. Following the tumor-resection-and-relapse approach (see Fig. 1a and Supplementary Fig. 1c ), mice injected with either 4T1 (e) or C26m2 tumor cells (f) were treated with either an isotype control antibody, or a neutralizing antibody against TNF-α, TGF-β, or both (200 μg antibody per mouse treated three times a week) starting one week after surgery, for a period of one week. For 4T1 model, n=8 non-tumor bearing control mice, n=5 tumor bearing mice treated with isotype control, n=6 tumor bearing mice treated with TNF-α antibody, n=7 tumor bearing mice treated with TGF-β antibody, and n=3 tumor bearing mice treated with both TNF-α and TGF-β antibodies. For C26m2 model, n=5 non-tumor bearing control mice, n=6 tumor bearing mice treated with isotype control, n=4 tumor bearing mice treated with TNF-α antibody, n=3 tumor bearing mice treated with TGF-β antibody, and n=5 tumor bearing mice treated with both TNF-α and TGF-β antibodies. Error bars represent SEM and all data were represented by mean ± SEM. P values were determined by two-tailed, unpaired Student’s t test in (a, b) and with one-way ANOVA with post-hoc Tukey’s test in (d-f) with F value (DFn, DFb) for d F(9, 36) = 161.9, e F(4, 24) = 10.3, and f F(4, 18) = 20.7. n.s., not significant. * P

    Journal: Nature medicine

    Article Title: Metastatic cancers promote cachexia through altered zinc homeostasis in skeletal muscle

    doi: 10.1038/s41591-018-0054-2

    Figure Lengend Snippet: ZIP14 is upregulated in cachectic muscles from metastatic mouse models and patients, and is induced by TNF-α and TGF-β (a,b) Body weight measurements (a) and qRT-PCR analysis of Trim63 , MAFbx , Fbxo31 , Musa1 and Zip14 in TA muscles (b) derived from four independent metastatic lung cancer models, compared to respective age-matched controls. Metastatic models include conditional Kras/p53 mutant ( Kras LSL-G12D/+ - p53 fl/fl ) and Kras/Lkb1 mutant ( Kras LSL-G12D/+ - Lkb1 fl/fl ) in which muscles were collected at 13 weeks post adeno-Cre induction, PC9-BrM3 xenograft in which muscles were collected at 7 weeks post tumor-cell injection, and Rb/p53 mutant allografts in which muscles were collected at 6 weeks post tumor resection (see also Supplementary Fig. 3a ). For body weight analysis in (a), n=4 controls and n=8 Kras/p53 conditional mutant mice, n=3 controls and n=6 Kras/Lkb1 conditional mutant mice, n=3 controls and n=10 PC9-BrM3 xenograft mice, and n=3 controls and n=10 Rb/p53 mutant allograft mice. For qRT-PCR analysis in (b), n=6 controls and n=4 Kras/p53 conditional mutant mice, n=3 controls and n=4 Kras/Lkb1 conditional mutant mice, n=4 controls and n=5 PC9-BrM3 xenograft mice, and n=5 controls and n=4 Rb/p53 mutant allograft model mice. (c) Representative images of ZIP14 immunohistochemistry on human muscle cross-sections from non-cachectic (upper panel) and cachectic (lower panel) metastatic cancer patients. ZIP14 antibody details are shown in Supplementary Fig. 3f-h . Scale bars, 50 μm. ca, cancer. Patient details are listed in Supplementary Table 3 . Data is representative of three independent experiments. (d) qRT-PCR analysis of Zip14 in human skeletal primary muscle cells treated with either vehicle, TNF-α (50 ng/ml), TGF-β (10 ng/ml), or both TNF-α (50 ng/ml) + TGF-β (10 ng/ml), either alone (vehicle) or in the presence of 10 μM of the indicated inhibitors. Cells were pretreated with either vehicle or the indicated inhibitors for 1 hour prior to adding the cytokines (TGF-β for 9 hours, and TNF-α for 3 hours before harvest). Cells from all groups were harvested at the same time. n=6 samples for cells treated with vehicle, TGF-β alone or TNF-α together with TGFβ; n=4 samples for all the other groups. Inhibitors: NF-κBi = NF-κB inhibitor (BAY 11-7085); AP1i = AP1 inhibitor (CC401); Smadi = TGFβ receptor I inhibitor (SB431542). (e,f) qRT-PCR analysis of Zip14 in TA muscles after neutralizing antibody treatment. Following the tumor-resection-and-relapse approach (see Fig. 1a and Supplementary Fig. 1c ), mice injected with either 4T1 (e) or C26m2 tumor cells (f) were treated with either an isotype control antibody, or a neutralizing antibody against TNF-α, TGF-β, or both (200 μg antibody per mouse treated three times a week) starting one week after surgery, for a period of one week. For 4T1 model, n=8 non-tumor bearing control mice, n=5 tumor bearing mice treated with isotype control, n=6 tumor bearing mice treated with TNF-α antibody, n=7 tumor bearing mice treated with TGF-β antibody, and n=3 tumor bearing mice treated with both TNF-α and TGF-β antibodies. For C26m2 model, n=5 non-tumor bearing control mice, n=6 tumor bearing mice treated with isotype control, n=4 tumor bearing mice treated with TNF-α antibody, n=3 tumor bearing mice treated with TGF-β antibody, and n=5 tumor bearing mice treated with both TNF-α and TGF-β antibodies. Error bars represent SEM and all data were represented by mean ± SEM. P values were determined by two-tailed, unpaired Student’s t test in (a, b) and with one-way ANOVA with post-hoc Tukey’s test in (d-f) with F value (DFn, DFb) for d F(9, 36) = 161.9, e F(4, 24) = 10.3, and f F(4, 18) = 20.7. n.s., not significant. * P

    Article Snippet: RNA extraction for RNA sequencing and qRT-PCR Total RNA was extracted using Trizol® reagent (Thermo Fisher) as previously described .

    Techniques: Quantitative RT-PCR, Derivative Assay, Mutagenesis, Injection, Mouse Assay, Immunohistochemistry, Two Tailed Test

    ZIP14-mediated zinc uptake in muscles promotes metastatic-cancer-induced cachexia (a,b) Representative immunofluorescence images (a) and associated morphometric analysis (b) of cross- sections of gastrocnemius muscles harvested from mice at 5 weeks post 4T1-cell injection. (a) Sections immunostained with antibody against laminin (shown in green) and stained with DAPI (shown in blue). Scale bars, 50 μm. (b) Morphometric analysis is depicted as the distribution frequency of fiber size categorized by fiber diameter. n=4 WT control mice, and n=3 mice for all other groups. (c) qRT-PCR analysis of the indicated atrophy markers in gastrocnemius, tibialis anterior or diaphragm muscles from Zip14 wild-type (WT) and Zip14 -knockout (KO) mice, with or without 4T1 tumor-cell injection, collected five weeks post injection. For gastrocnemius muscles, n=7 WT controls, n=5 WT mice bearing 4T1, n=6 KO mice and n=7 KO mice bearing 4T1 for analyzing Trim63 expression; n=7 WT controls, n=6 WT mice bearing 4T1, n=6 KO mice and n=8 KO mice bearing 4T1 for MAFbx expression; n=7 WT controls, n=5 WT mice bearing 4T1, n=6 KO mice and n=9 KO mice for Fbxo31 expression, n=7 WT controls, n=7 WT mice bearing 4T1, n=6 KO mice and n=10 KO mice bearing 4T1 for Musa1 expression. For TA muscles, n=7 WT controls, n=6 WT mice bearing 4T1, n=5 KO mice and n=7 KO mice bearing 4T1 for Trim63 and MAFbx expression; n=5 WT controls, n=5 WT mice bearing 4T1, n=5 KO mice and n=8 KO mice for Fbxo31 expression, n=5 WT controls, n=6 WT mice bearing 4T1, n=5 KO mice and n=8 KO mice bearing 4T1 for Musa1 expression. For diaphragm muscles, n=3 per group. Data were normalized to non-tumor- bearing Zip14 WT mice. (d) AAV vectors expressing mCherry and a shRNA targeting either a scrambled sequence (shCon) or Zip14 (sh Zip14 ) were injected intramuscularly into the gastrocnemius muscles and monitored by fluorescence imaging. A representative image taken five weeks after injection of AAV particles intramuscularly is shown in the upper panel. C26m2 cancer cells were then subcutaneously injected, and metastasis was monitored as previously described (see Fig. 1a and Supplementary Fig. 1c ). Muscles were collected five weeks after tumor-cell injection, and Zip14 expression was determined by qRT-PCR analysis (lower panel). n=4 mice per group. Data were normalized to shCon. (e,f) Representative immunofluorescence staining images of laminin (e) and morphometric analysis of muscle size in gastrocnemius muscles from C26m2 tumor-bearing (Tb) mice injected with either shCon or sh Zip14 (f). (e) Sections immunostained with antibody against laminin (shown in green) and stained with DAPI (shown in blue). Scale bars, 50 μm. (f) Morphometric analysis is depicted as the distribution frequency of fiber size categorized by fiber diameter. n=3 mice for shCon, and n=4 mice for sh Zip14 . (g) qRT-PCR analysis of the indicated genes in gastrocnemius muscles from C26m2 tumor-bearing (Tb) mice shown in (d), n=4 mice per group. Data were normalized to shCon. (h) Zinc levels in gastrocnemius (gast), TA and diaphragm (dia) muscles (μg/g of dry weight) determined by inductively-coupled-plasma-mass-spectrometry (ICP/MS) analysis from either non-tumor-bearing control mice or mice bearing either 4T1 or C26m2 metastases collected at 5 weeks post tumor-cell injection. For 4T1 model, n=7 controls and n=8 tumor bearing mice for gastrocnemius muscles; n=4 controls and n=6 tumor bearing mice for TA muscles; n=3 mice per group for diaphragm muscles. For C26m2 model, n=7 mice per group for gastrocnemius muscles; n=3 mice per group for TA muscles; n=8 controls and n=10 tumor bearing mice for diaphragm muscles. (i,j) Body weight analysis (i) and qRT-PCR analysis of the indicated genes in TA muscles (j) of Zip14 wild-type (WT) and Zip14 knockout (KO) mice, either injected with C26m2 cancer cells or left uninjected as non-tumor-bearing controls. Mice were subdivided into two groups on the day of tumor cell injection and treated with either normal or zinc-supplemented drinking water for the indicated number of days in (i). TA muscles were harvested for qRT-PCR analysis after 15 days on zinc-supplemented water in (j). For body weight analysis (i), n=8 mice for WT, Tb-WT and Tb-WT+Zn; n=7 mice for WT+Zn; n=4 mice for KO, KO+Zn and Tb-KO; n=5 for Tb-KO+Zn. For gene expression analysis (j), n=5 WT mice, n=3 WT+Zn mice, n=3 Tb-WT mice, n=7 Tb-WT+Zn mice, n=3 KO mice, n=3 KO+Zn mice, n=3 Tb-KO mice and n=4 Tb-KO+Zn mice for analyzing Trim63 and MAFbx ; n=5 WT mice, n=3 WT+Zn mice, n=3 Tb-WT mice, n=7 Tb-WT+Zn, n=4 KO mice, n=3 KO+Zn mice, n=4 Tb-KO mice and n=4 Tb-KO+Zn mice for analyzing Fbxo31 and Musa1 . Data in (j) were normalized to non-tumor- bearing Zip14 WT mice on regular water without zinc supplementation. Error bars represent SEM and all data were represented by mean ± SEM. P values were determined by two-tailed, unpaired Student’s t-test in (d, g, h and i), two-sided Welch’s t-test in (b and f), and one-way ANOVA with post-hoc Tukey’s test in (c and j) with F value (DFn, DFd) for c Trim63 -Gastrocnemius F(3, 21) = 221.0, c Trim63 -TA F(3, 21) = 13.7, c Trim63 -Diaphragm F(3, 8) = 39.1, c MAFbx -Gastrocnemius F(3, 23) = 101.0, c MAFbx -TA F(3, 21) = 10.4, c MAFbx -Diaphragm F(3, 8) = 17.3, c Fbxo31 -Gastrocnemius F(3, 23) = 41.2, c Fbxo31 -TA F(3, 19) = 11.6, c Fbxo31 -Diaphragm F(3, 8) = 16.3, c Musa1 -Gastrocnemius F(3, 26) = 12.3, c Musa1 -TA F(3, 20) = 22.2, c Musa1 -Diaphragm F(3, 8) = 11.2, j Trim63 F(7, 23) = 8.5, j MAFbx F(7, 23) = 10.0, j Fbxo31 F(7, 25) = 51.5, j Musa1 F(7, 25) = 10.7. n.s., not significant. ** P

    Journal: Nature medicine

    Article Title: Metastatic cancers promote cachexia through altered zinc homeostasis in skeletal muscle

    doi: 10.1038/s41591-018-0054-2

    Figure Lengend Snippet: ZIP14-mediated zinc uptake in muscles promotes metastatic-cancer-induced cachexia (a,b) Representative immunofluorescence images (a) and associated morphometric analysis (b) of cross- sections of gastrocnemius muscles harvested from mice at 5 weeks post 4T1-cell injection. (a) Sections immunostained with antibody against laminin (shown in green) and stained with DAPI (shown in blue). Scale bars, 50 μm. (b) Morphometric analysis is depicted as the distribution frequency of fiber size categorized by fiber diameter. n=4 WT control mice, and n=3 mice for all other groups. (c) qRT-PCR analysis of the indicated atrophy markers in gastrocnemius, tibialis anterior or diaphragm muscles from Zip14 wild-type (WT) and Zip14 -knockout (KO) mice, with or without 4T1 tumor-cell injection, collected five weeks post injection. For gastrocnemius muscles, n=7 WT controls, n=5 WT mice bearing 4T1, n=6 KO mice and n=7 KO mice bearing 4T1 for analyzing Trim63 expression; n=7 WT controls, n=6 WT mice bearing 4T1, n=6 KO mice and n=8 KO mice bearing 4T1 for MAFbx expression; n=7 WT controls, n=5 WT mice bearing 4T1, n=6 KO mice and n=9 KO mice for Fbxo31 expression, n=7 WT controls, n=7 WT mice bearing 4T1, n=6 KO mice and n=10 KO mice bearing 4T1 for Musa1 expression. For TA muscles, n=7 WT controls, n=6 WT mice bearing 4T1, n=5 KO mice and n=7 KO mice bearing 4T1 for Trim63 and MAFbx expression; n=5 WT controls, n=5 WT mice bearing 4T1, n=5 KO mice and n=8 KO mice for Fbxo31 expression, n=5 WT controls, n=6 WT mice bearing 4T1, n=5 KO mice and n=8 KO mice bearing 4T1 for Musa1 expression. For diaphragm muscles, n=3 per group. Data were normalized to non-tumor- bearing Zip14 WT mice. (d) AAV vectors expressing mCherry and a shRNA targeting either a scrambled sequence (shCon) or Zip14 (sh Zip14 ) were injected intramuscularly into the gastrocnemius muscles and monitored by fluorescence imaging. A representative image taken five weeks after injection of AAV particles intramuscularly is shown in the upper panel. C26m2 cancer cells were then subcutaneously injected, and metastasis was monitored as previously described (see Fig. 1a and Supplementary Fig. 1c ). Muscles were collected five weeks after tumor-cell injection, and Zip14 expression was determined by qRT-PCR analysis (lower panel). n=4 mice per group. Data were normalized to shCon. (e,f) Representative immunofluorescence staining images of laminin (e) and morphometric analysis of muscle size in gastrocnemius muscles from C26m2 tumor-bearing (Tb) mice injected with either shCon or sh Zip14 (f). (e) Sections immunostained with antibody against laminin (shown in green) and stained with DAPI (shown in blue). Scale bars, 50 μm. (f) Morphometric analysis is depicted as the distribution frequency of fiber size categorized by fiber diameter. n=3 mice for shCon, and n=4 mice for sh Zip14 . (g) qRT-PCR analysis of the indicated genes in gastrocnemius muscles from C26m2 tumor-bearing (Tb) mice shown in (d), n=4 mice per group. Data were normalized to shCon. (h) Zinc levels in gastrocnemius (gast), TA and diaphragm (dia) muscles (μg/g of dry weight) determined by inductively-coupled-plasma-mass-spectrometry (ICP/MS) analysis from either non-tumor-bearing control mice or mice bearing either 4T1 or C26m2 metastases collected at 5 weeks post tumor-cell injection. For 4T1 model, n=7 controls and n=8 tumor bearing mice for gastrocnemius muscles; n=4 controls and n=6 tumor bearing mice for TA muscles; n=3 mice per group for diaphragm muscles. For C26m2 model, n=7 mice per group for gastrocnemius muscles; n=3 mice per group for TA muscles; n=8 controls and n=10 tumor bearing mice for diaphragm muscles. (i,j) Body weight analysis (i) and qRT-PCR analysis of the indicated genes in TA muscles (j) of Zip14 wild-type (WT) and Zip14 knockout (KO) mice, either injected with C26m2 cancer cells or left uninjected as non-tumor-bearing controls. Mice were subdivided into two groups on the day of tumor cell injection and treated with either normal or zinc-supplemented drinking water for the indicated number of days in (i). TA muscles were harvested for qRT-PCR analysis after 15 days on zinc-supplemented water in (j). For body weight analysis (i), n=8 mice for WT, Tb-WT and Tb-WT+Zn; n=7 mice for WT+Zn; n=4 mice for KO, KO+Zn and Tb-KO; n=5 for Tb-KO+Zn. For gene expression analysis (j), n=5 WT mice, n=3 WT+Zn mice, n=3 Tb-WT mice, n=7 Tb-WT+Zn mice, n=3 KO mice, n=3 KO+Zn mice, n=3 Tb-KO mice and n=4 Tb-KO+Zn mice for analyzing Trim63 and MAFbx ; n=5 WT mice, n=3 WT+Zn mice, n=3 Tb-WT mice, n=7 Tb-WT+Zn, n=4 KO mice, n=3 KO+Zn mice, n=4 Tb-KO mice and n=4 Tb-KO+Zn mice for analyzing Fbxo31 and Musa1 . Data in (j) were normalized to non-tumor- bearing Zip14 WT mice on regular water without zinc supplementation. Error bars represent SEM and all data were represented by mean ± SEM. P values were determined by two-tailed, unpaired Student’s t-test in (d, g, h and i), two-sided Welch’s t-test in (b and f), and one-way ANOVA with post-hoc Tukey’s test in (c and j) with F value (DFn, DFd) for c Trim63 -Gastrocnemius F(3, 21) = 221.0, c Trim63 -TA F(3, 21) = 13.7, c Trim63 -Diaphragm F(3, 8) = 39.1, c MAFbx -Gastrocnemius F(3, 23) = 101.0, c MAFbx -TA F(3, 21) = 10.4, c MAFbx -Diaphragm F(3, 8) = 17.3, c Fbxo31 -Gastrocnemius F(3, 23) = 41.2, c Fbxo31 -TA F(3, 19) = 11.6, c Fbxo31 -Diaphragm F(3, 8) = 16.3, c Musa1 -Gastrocnemius F(3, 26) = 12.3, c Musa1 -TA F(3, 20) = 22.2, c Musa1 -Diaphragm F(3, 8) = 11.2, j Trim63 F(7, 23) = 8.5, j MAFbx F(7, 23) = 10.0, j Fbxo31 F(7, 25) = 51.5, j Musa1 F(7, 25) = 10.7. n.s., not significant. ** P

    Article Snippet: RNA extraction for RNA sequencing and qRT-PCR Total RNA was extracted using Trizol® reagent (Thermo Fisher) as previously described .

    Techniques: Immunofluorescence, Mouse Assay, Injection, Staining, Quantitative RT-PCR, Knock-Out, Expressing, shRNA, Sequencing, Fluorescence, Imaging, Mass Spectrometry, Two Tailed Test

    Morrbid represses Bcl2l11 by maintaining its bivalent promoter in a poised state and Morrbid -haploinsufficiency (a) Venn diagram summary of EZH2 PAR-CLIP analysis, with representation of tags and RNA-protein contact sites (RCSs) as determined by PARalyzer mapping to Morrbid. (b) Co-immunoprecipitation of the PRC2 family member EZH2 and Morrbid . Nuclear extracts of immortalized WT bone marrow-derived macrophages (BMDMs) stimulated with LPS for 6-12 hours were immunoprecipitated by IgG or anti-EZH2. Co-precipitation of indicated RNAs were assayed by qPCR. Data are represented as enrichment over IgG control (n=6 biological replicates pooled from 2 independent experiments, representative of 3 independent experiments). (c) Validation of Morrbid RNA pull-down over other RNAs using pools of Morrbid capture probes and LacZ probes (n=3, average of 3 independent experiments). (d) Visualized 3C PCR products from bait and indicated reverse primers using template from fixed and ligated bone marrow derived eosinophil DNA (S1, S2, and S3), BAC control (BAC), or water. The sequence of each reverse primer is listed in Supplementary Table 1 . (e-f) Bone marrow-derived eosinophils from wild-type (WT) and Bcl2l11−/− mice treated with EZH2 inhibitor GSK126 over time. (e) Frequency of non-viable (Aqua+) and (f) annexin V staining cells on day 5 following treatment with GSK126 (n=3 independently differentiated eosinophils per dose, results representative of 2 independent experiments). (g) (Top) Total cell numbers and (Bottom) BCL2L11 protein expression of indicated cell populations from the blood of wild-type (WT), Morrbid -heterozygous, and Morrbid- deficient mice (n=3-5 mice per group, results representative of 3 independent experiments). Error bars show s.e.m. *p

    Journal: Nature

    Article Title: The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan

    doi: 10.1038/nature19346

    Figure Lengend Snippet: Morrbid represses Bcl2l11 by maintaining its bivalent promoter in a poised state and Morrbid -haploinsufficiency (a) Venn diagram summary of EZH2 PAR-CLIP analysis, with representation of tags and RNA-protein contact sites (RCSs) as determined by PARalyzer mapping to Morrbid. (b) Co-immunoprecipitation of the PRC2 family member EZH2 and Morrbid . Nuclear extracts of immortalized WT bone marrow-derived macrophages (BMDMs) stimulated with LPS for 6-12 hours were immunoprecipitated by IgG or anti-EZH2. Co-precipitation of indicated RNAs were assayed by qPCR. Data are represented as enrichment over IgG control (n=6 biological replicates pooled from 2 independent experiments, representative of 3 independent experiments). (c) Validation of Morrbid RNA pull-down over other RNAs using pools of Morrbid capture probes and LacZ probes (n=3, average of 3 independent experiments). (d) Visualized 3C PCR products from bait and indicated reverse primers using template from fixed and ligated bone marrow derived eosinophil DNA (S1, S2, and S3), BAC control (BAC), or water. The sequence of each reverse primer is listed in Supplementary Table 1 . (e-f) Bone marrow-derived eosinophils from wild-type (WT) and Bcl2l11−/− mice treated with EZH2 inhibitor GSK126 over time. (e) Frequency of non-viable (Aqua+) and (f) annexin V staining cells on day 5 following treatment with GSK126 (n=3 independently differentiated eosinophils per dose, results representative of 2 independent experiments). (g) (Top) Total cell numbers and (Bottom) BCL2L11 protein expression of indicated cell populations from the blood of wild-type (WT), Morrbid -heterozygous, and Morrbid- deficient mice (n=3-5 mice per group, results representative of 3 independent experiments). Error bars show s.e.m. *p

    Article Snippet: RNA extraction, cDNA synthesis, and quantitative RT-PCR Total RNA was extracted from TRIzol (Life Technologies) according to the manufacturer's instructions.

    Techniques: Cross-linking Immunoprecipitation, Immunoprecipitation, Derivative Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, BAC Assay, Sequencing, Mouse Assay, Staining, Expressing

    AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

    Journal: American Journal of Translational Research

    Article Title: Two-layer regulation of TRAF6 mediated by both TLR4/NF-kB signaling and miR-589-5p increases proinflammatory cytokines in the pathology of severe acute pancreatitis

    doi:

    Figure Lengend Snippet: AZA treatment affected TRAF6 downstream signaling. (A) AZA treatment affected the protein levels of TRAF6 downstream members. Total cell extracts from PPAC cells transfected with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) AZA treatment had no effect on NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were subjected to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in cytoplasmic fractions. The cytoplasmic fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions were significantly decreased after AZA treatment. The nuclear fractions from PPAC cells treated with different concentrations of AZA (0, 2.5 and 25 mM) were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) AZA treatment decreased the expression of NF-kB targets. The cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

    Article Snippet: Total RNA isolation and qRT-PCR analysisThe cultured cells and pancreatic tissues were subjected to total RNA isolation with a TRIzol reagent (Thermo Fisher Scientific, #15596026) following a method provided by the manufacturer.

    Techniques: Transfection, Expressing, Isolation, Quantitative RT-PCR

    Overexpression or downregulation of miR-589-5p affected the TLR4/NF-kB signaling. (A) Changes in miR-589-5p levels affected the protein levels of several members of the TLR4/NF-kB signaling pathway. Total cell extracts from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) Changes in miR-589-5p levels did not affect NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were applied to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in the cytoplasmic fractions. The cytoplasmic fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions. The nuclear fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) Changes in miR-589-5p levels affected the expression of NF-kB targets. Cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

    Journal: American Journal of Translational Research

    Article Title: Two-layer regulation of TRAF6 mediated by both TLR4/NF-kB signaling and miR-589-5p increases proinflammatory cytokines in the pathology of severe acute pancreatitis

    doi:

    Figure Lengend Snippet: Overexpression or downregulation of miR-589-5p affected the TLR4/NF-kB signaling. (A) Changes in miR-589-5p levels affected the protein levels of several members of the TLR4/NF-kB signaling pathway. Total cell extracts from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine the protein levels of TLR4, TRAF6, IKK1, IkB and pIkB. GAPDH was used as a loading control. (B) Changes in miR-589-5p levels did not affect NF-kB subunit protein levels in total extracts. The same cell extracts used in (A) were applied to immunoblot analyses to examine the protein levels of RELA, RELB, c-REL, NFKB1 and NFKB2. GAPDH was used as a loading control. (C) The protein levels of NF-kB subunits in the cytoplasmic fractions. The cytoplasmic fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. β-Actin was used as a loading control. (D) The protein levels of NF-kB subunits in nuclear fractions. The nuclear fractions from MIA PaCa-2 cells transfected with miR-NC, miR-589-5p-mimic and anti-miR-589-5p were subjected to immunoblot analyses to examine NF-kB subunit protein levels. LSD1 was used as a loading control. (E) Changes in miR-589-5p levels affected the expression of NF-kB targets. Cells used in (A) were used for RNA isolation and qRT-PCR analyses to measure the expression of NF-kB targets, including IL-1B, IL-6, IL-8, TNFA, IFNB1, CCL3 and LTA . ** P

    Article Snippet: Total RNA isolation and qRT-PCR analysisThe cultured cells and pancreatic tissues were subjected to total RNA isolation with a TRIzol reagent (Thermo Fisher Scientific, #15596026) following a method provided by the manufacturer.

    Techniques: Over Expression, Transfection, Expressing, Isolation, Quantitative RT-PCR