Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Dendritic cells in the bladder microenvironment of mice responding to CD40 agonism have higher expression of IL-15Rα. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 24 post-tumor implantation (n = 3-6 mice per group; bars represent SD). Responders (gray circles) and non-responders (blue circles) to anti-CD40 antibody therapy are indicated. ( C ) IL-15Rα mean fluorescence intensity across mice on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 24 post-tumor implantation (n = 3-6 mice per group). *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Luciferase, Imaging, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Endogenous IL-15 participates in the therapeutic activity of CD40 agonism. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody and/or anti-IL-15 blocking antibody or isotype-matched control antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 12 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 5 mice per group) of CD44 hi CD122 + CD8 T cells (top) and CXCR6 + CD44 hi CD122 + CD8 T cells (bottom) in the bladders of mice treated as outlined in A. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Blocking Assay, Control, Luciferase, Imaging, Tumor Implantation, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: DCs in human non-muscle invasive bladder cancer express high levels of IL-15. Eight primary tumor samples from either untreated NMIBC patients (n=3) or from NMIBC patients that had progressed from NMIBC to MIBC following BCG therapy (n=5) were included in this single-cell RNA sequencing analysis. A) Phenograph clustering was performed and 29 clusters were identified. Canonical lineage markers for cell populations were utilized to assign cell identities to each cluster. B) tSNE plots were created for four representative markers: CD40, CD40L, IL15, and IL15RA. C) A matrix plot with column scaled expression of the same four markers of interest is shown to highlight the distribution and intensity of expression for each gene across each subset.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15/IL-15Rα upregulation on dendritic cells in the bladder tumor microenvironment. Mice bearing orthotopic MB49 bladder tumors were treated intravesically with anti-CD40 antibody, BCG, or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). ( C ) Representative flow cytometry plots and ( D ) quantification across mice of IL-15 and IL-15Rα surface expression on cDC1s in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). Left graph is gated on cDC1s and depicts proportions of cDC1s that are double-positive for surface IL-15 and IL-15Rα. Right graph is gated on IL-15Rα-expressing cDC1s and depicts proportions of cDC1s expressing IL-15Rα that is occupied by IL-15. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15Rα upregulation on dendritic cells in the UPPL1541 bladder tumor microenvironment. Mice bearing orthotopic UPPL1541 bladder tumors were treated intravesically with anti-CD40 antibody or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances primary anti-tumor activity. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative intravital luciferase imaging (left) and luminescence quantification (right) across mice at day 14 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Survival (top) and representative intravital luciferase imaging (bottom) of surviving mice at day 85 post-tumor implantation treated as outlined in A. ( D ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across mice treated with anti-CD40 antibody 2141-V11 and IL-15 or control in the absence or presence of additional anti-CD8 T cell depleting antibody (n = 5 mice per group; bars represent SD). ( E ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across WT or Batf3 -/- mice treated with anti-CD40 antibody 1C10 and IL-15 or control (n = 5 mice per group; bars represent SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Luciferase, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination of Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor activity against UPPL1541 tumors. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic UPPL1541 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative bladder ultrasound imaging (left) at day 45 post-tumor implantation and disease-free survival (right) of mice treated as outlined in A. *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: IL-15 therapy does not modulate myeloid CD40 expression. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with IL-15 or vehicle control. ( B ) Bladder weights across mice at day 12 post-tumor implantation (n = 4-5 mice per group; bars represent SD). ( C ) CD40 mean fluorescence intensity across mice on total dendritic cells (DC; defined as F4/80 − Ly-6G − CD11c + MHCII + ), type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), monocytes (defined as CD11b hi Ly6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 4-5 mice per group). ns = not significant.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor memory responses. ( A ) Schematic of subcutaneous rechallenge (ten-fold tumor cell dose) of humanized hCD40/hFcyR mice surviving long-term (>90 days after initial orthotopic MB49 bladder tumor implantation) following initial therapy with anti-CD40 antibody 2141-V11 with or without IL-15. ( B ) Tumor growth (in the absence of any additional therapy) of long-term survivors compared with naïve mice receiving an equivalent MB49 tumor cell implant (n = 2-5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 2-5 mice per group) of CD44 hi CD122 + CD8 T cells (top), CD44 hi CD122 + CD62L + CD8 T cells (middle), and CD11b + KLRG1 + CD27 − NK cells (bottom) in the peripheral blood of mice surviving long-term (day 110) after primary tumor implantation and tumor rechallenge. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Tumor Implantation, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism

doi: 10.1007/s00018-022-04680-w

Figure Lengend Snippet: FHL3 directly interacts with ZN2 domain of YY1 and decreases its binding capacities to MyHC2b gene regulatory region. A C2C12 myoblasts were co-transfected with pcDNA3.1-FHL3 and YY1 siRNA (indicated at the bottom) and differentiated for 4 days. The cell lysates were subject to western blotting with anti-FHL3, anti-YY1 and anti-MyHC2b (indicated at the left). Western blotting results showed that knockdown of YY1 could reduce the promotion effect of FHL3 overexpression on MyHC2b expression. The relative protein levels were normalized to β-actin. The data were presented as mean ± SD of three independent experiments; *P < 0.05, **P < 0.01. B C2C12 myoblasts were co-transfected with FHL3 siRNA and pcDNA3.1-YY1, and then differentiated for 4 days. The cell lysates were subject to western blotting with anti-FHL3, anti-YY1, and anti-MyHC2b. Western blotting results showed that FHL3 knockdown could increase the inhibitory effects of YY1 overexpression on MyHC2b expression. The relative protein levels were normalized to β-actin. The data were presented as mean ± SD of three independent experiments; *P < 0.05, ***P < 0.001. C Immunofluorescence staining showed that FHL3 protein co-located with YY1 in cell nucleus of C2C12 myoblasts differentiated for 2 days. C2C12 myoblasts were stained with anti-FHL3 (red), anti-YY1 (green), and DAPI (blue), and were imaged by confocal laser scanning microscopy. Scale bars, 5 μm. D, E The co-immunoprecipitation (Co-IP) results showing the interaction of FHL3 and YY1 in vivo. C2C12 myoblasts were differentiated for 2 days, harvested and co-immunoprecipitated with anti-FHL3 or anti-IgG (D) and with anti-IgG or anti-YY1 (E). IgG was used as a negative control. The total cell lysates before immunoprecipitation were used as input to verify expression of FHL3 and YY1. F Schematic diagram of different truncated fragments of FHL3 (up) and GST pulldown assays. In vitro translated proteins of GST, GST-FHL3-LIM1/2 (1–39), GST-FHL3-LIM1 (40–98), GST-FHL3-LIM2 (99–159), GST-FHL3-LIM3 (160–218), GST-FHL3-LIM4 (219–289) with His-YY1 were used for GST pulldown assays. The GST pulldown results showed GST-FHL3 and each LIM domain of FHL3 could directly interact with His-YY1 in vitro (up), GST-tagged proteins were detected by western blotting with anti-GST (down). G Schematic diagram of different truncated fragments of YY1. YY1 functional domains include transactivation domain (YY1-TD; 1–99), repression domain (YY1-RD; 170–225), and DNA-binding domain (YY1-DD; 294–414). YY1-RD domain includes HAT-HDAC interaction domain (YY1-RD1; 170–200) and REPO domain (YY1-RD2; 200–225); YY1-DD domain includes four zinc finger domains (ZN): ZN1 (294–322), ZN2 (323–350), ZN3 (351–380) and ZN4 (381–414), respectively. H GST pulldown results indicated that GST-YY1-RD and GST-YY1-DD could interact with His-FHL3. In vitro translated proteins of GST, GST-YY1-TD, GST-YY1-RD, GST-YY1-DD, GSY-YY1, and His-FHL3 were used for GST pulldown assays. The interaction of GST-YY1 truncated proteins with His-FHL3 was detected by western blotting with anti-His (up). GST-tagged proteins were detected by western blotting with anti-GST (down). I GST pulldown indicated that GST-YY1-ZN2 could interact with His-FHL3. In vitro translated proteins of GST, GST-YY1-ZN1, GST-YY1-ZN2, GST-YY1-ZN3, GST-YY1-ZN4, GST-YY1-DD, and His-FHL3 were used for GST pulldown analysis. The interaction of GST-YY-DD truncated proteins with His-FHL3 was detected by western blotting with anti-His (up). GST-tagged proteins were detected by western blotting with anti-GST (down). J EMSA experiments were used to analyze the binding of GST, GST-YY1-ZN1, GST-YY1-ZN2, GST-YY1-ZN3, GST-YY1-ZN4, and GST-YY1-DD fusion proteins to MyHC2b regulatory regions in vitro. GST, and free H2O were used for negative control and blank control, respectively. The specific DNA–protein complex band was observed in the wild-type probes incubated with GST-YY1-ZN1, GST-YY1-ZN2, GST-YY1-ZN3, and GST-YY1-DD. K ChIP-qPCR results showed the YY1 enrichments at the MyHC2b regulatory regions were significantly decreased after FHL3 overexpression in C2C12 myoblasts differentiated for 2 days. IgG was performed as controls, and the precipitated DNA was amplified by PCR with primers for the MyHC2b gene regulatory region. *P < 0.05. L EMSA results showing the effect of the FHL3 on the binding capacity of YY1-DD to the MyHC 2b gene regulatory region in vitro. GST, and free H2O were used as negative and blank control, respectively. A specific DNA–protein complex band was observed in the wild-type probes incubated with GST-YY1-DD, while the DNA–protein complex band became weaker and a super band formed when His-FHL3 was added

Article Snippet: Mouse FHL3 small interfering RNA (siRNA) oligonucleotides (sense: CCAUGAGCGAGGCAUUUGATT; anti-sense: UCAAAUGCCUCGCUCAUGGTT); mouse YY1 siRNA oligonucleotides (sense: GGACCUUUACUGCCACAAATT; anti-sense: UUUGUGGCAGUAAAGGUCCTT) and pig FHL3 siRNA Oligonucleotides (siRNA1-sense:AGCGCAAAUACAUUCAGACGTT; siRNA1-anti-sense:CGUCUGAAUGUAUUUGCGCTT; siRNA2-sense:GCUCUGUAACGACUGCUACTT; siRNA2-anti-sense:GUAGCAGUCGUUACAGAGCTT; siRNA3-sense:CCGGGACGAUGAUCCUUAUTT; siRNA3-anti-sense: AUAAGGAUCAUCGUCCCGGTT) were designed and synthesized by GenePharma (China, Shanghai).

Techniques: Binding Assay, Transfection, Western Blot, Knockdown, Over Expression, Expressing, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Negative Control, In Vitro, Functional Assay, Control, Incubation, ChIP-qPCR, Amplification

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism

doi: 10.1007/s00018-022-04680-w

Figure Lengend Snippet: Bioinformatics analysis of differentially expressed genes between WT and FHL3 knockout C2C12 cells revealed that YY1 may participate in the regulation of the formation of fast glycolytic muscle fibers by FHL3. A Western blotting results showed that FHL3 knockout significantly decreased the expression levels of MyHC2a and MyHC2b, but significantly increased the expression levels of MyHC1/slow in C2C12 cells differentiated for 4 days. B Volcano plot of differentially expressed genes (DEGs) between WT and FHL3 KO C2C12 cells. Blue: downregulated DEGs, red: upregulated DEGs. C GO enrichment dot plot of the differentially upregulated genes after FHL3 knockout. D Gene set enrichment analysis (GSEA) of DEGs related to ‘transition between fast and slow fiber.’ E Heatmap of differentially expressed fast-twitch and slow-twitch related genes. F qRT-PCR results showed that the expression levels of fast-switch related genes were significantly decreased, and the expression levels of slow-switch genes were significantly increased in FHL3 knockout cells compared with WT cells. G Veen diagram showing that YY1 was the common gene between 12 potential transcription factors that specifically bind to MyHC2b regulatory region by PROMO analysis and 87 regulatory factors predicted by RNA-seq DEGs BART analysis. The relative protein and mRNA levels were normalized to β-actin. The data were presented as mean ± SD of three independent experiments; P values were determined by paired t test. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Mouse FHL3 small interfering RNA (siRNA) oligonucleotides (sense: CCAUGAGCGAGGCAUUUGATT; anti-sense: UCAAAUGCCUCGCUCAUGGTT); mouse YY1 siRNA oligonucleotides (sense: GGACCUUUACUGCCACAAATT; anti-sense: UUUGUGGCAGUAAAGGUCCTT) and pig FHL3 siRNA Oligonucleotides (siRNA1-sense:AGCGCAAAUACAUUCAGACGTT; siRNA1-anti-sense:CGUCUGAAUGUAUUUGCGCTT; siRNA2-sense:GCUCUGUAACGACUGCUACTT; siRNA2-anti-sense:GUAGCAGUCGUUACAGAGCTT; siRNA3-sense:CCGGGACGAUGAUCCUUAUTT; siRNA3-anti-sense: AUAAGGAUCAUCGUCCCGGTT) were designed and synthesized by GenePharma (China, Shanghai).

Techniques: Knock-Out, Western Blot, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism

doi: 10.1007/s00018-022-04680-w

Figure Lengend Snippet: YY1 directly binds to MyHC2b gene regulatory region and specifically represses its gene expression. A Luciferase reporter assay in C2C12 cells differentiated for 2 days indicated that the FHL3 overexpression significantly increased the transcription activities of the D5, D6, and D7 truncated fragments containing two putative YY1 binding motifs. Left panel, schematic diagram of eight truncated fragments of MyHC2b gene regulatory region linked to luciferase gene in pGL3 vector. The nucleotides were numbered relative to the translation start site that was assigned as + 1. Schematic diagram of two potential YY1 binding motifs (A, B) in truncated fragments of MyHC2b gene regulatory region were displayed. Right panel, the relative activities of a series of truncated fragments of the pGL3-MyHC2b construct determined by luciferase assay after transfection of FHL3 overexpression vector (pcDNA3.1-FHL3). B Schematic diagram of two YY1 binding sites (A, B) in MyHC2b gene regulatory region between nucleotides − 1100 bp and − 910 bp which were predicted by PROMO and mutated motifs. The dual luciferase assays showed that overexpression of FHL3 did not significantly enhance the fluorescence activity of MyHC2b D5 truncated fragment only when motif A was mutated. C ChIP-qPCR results showed that YY1 could bind to MyHC2b gene regulatory region in C2C12 cells differentiated for 2 days. IgG was performed as controls and precipitated DNA was amplified by PCR with primers for the MyHC2b gene regulatory region. D EMSA assays were used to analyze the binding of GST, free H2O, and GST-YY1 fusion protein to MyHC2b regulatory regions in vitro. GST, and free H2O were used for negative control and blank control, respectively. The specific DNA–protein complex band was observed in the WT probes incubated with GST-YY1. E Representative images of MyHC1/slow, MyHC2a, and MyHC2b immunofluorescence staining and quantification in three independent experiments indicated that YY1 overexpression (pcDNA3.1-YY1) significantly decreased the proportion of MyHC2b-positive myotubes, and did not significantly affect the proportion of MyHC1/slow- and MyHC2a-positive myotubes in C2C12 cells differentiated for 4 days. The myotubes were stained with anti-MyHC1/slow antibodies (red), anti-MyHC2a antibodies (red), anti-MyHC2b antibodies (red), and DAPI (blue). Scale bars, 50 μm. F Western blotting results showed that YY1 overexpression significantly decreased the expression levels of MyHC2b, and had no significant effects on the expression levels of MyHC1/slow and MyHC2a in C2C12 cells differentiated for 4 days. The relative protein levels were normalized to β-actin. The data were presented as mean ± SD of three independent experiments; *P < 0.05, **P < 0.01, N.S. indicates statistical non-significance

Article Snippet: Mouse FHL3 small interfering RNA (siRNA) oligonucleotides (sense: CCAUGAGCGAGGCAUUUGATT; anti-sense: UCAAAUGCCUCGCUCAUGGTT); mouse YY1 siRNA oligonucleotides (sense: GGACCUUUACUGCCACAAATT; anti-sense: UUUGUGGCAGUAAAGGUCCTT) and pig FHL3 siRNA Oligonucleotides (siRNA1-sense:AGCGCAAAUACAUUCAGACGTT; siRNA1-anti-sense:CGUCUGAAUGUAUUUGCGCTT; siRNA2-sense:GCUCUGUAACGACUGCUACTT; siRNA2-anti-sense:GUAGCAGUCGUUACAGAGCTT; siRNA3-sense:CCGGGACGAUGAUCCUUAUTT; siRNA3-anti-sense: AUAAGGAUCAUCGUCCCGGTT) were designed and synthesized by GenePharma (China, Shanghai).

Techniques: Gene Expression, Luciferase, Reporter Assay, Over Expression, Binding Assay, Plasmid Preparation, Construct, Transfection, Fluorescence, Activity Assay, ChIP-qPCR, Amplification, In Vitro, Negative Control, Control, Incubation, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism

doi: 10.1007/s00018-022-04680-w

Figure Lengend Snippet: FHL3 reduces EZH2 recruitment to regulatory regions of MyHC2b by competitively interacting with YY1-RD2 domain. A GST pulldown results indicated that GST-YY1-RD1 and GST-YY1-RD2 could interact with His-FHL3 in vitro. In vitro translated proteins of GST, GST-YY1-RD1, GST-YY1-RD2, GST-YY1-RD, and His-FHL3 were used for GST pulldown assays. The interaction of GST-YY1 truncated proteins with His-FHL3 was detected by western blotting with anti-His (up). The GST-tagged proteins were detected by western blotting with anti-GST (down). B, C The Co-IP results showed that FHL3 overexpression (B) and knockdown (C) significantly decreased and increased the interaction of EZH2 with YY1 in vivo, respectively. C2C12 myoblasts differentiated for 2 days were co-immunoprecipitated with anti-YY1 or anti-IgG. The immunoprecipitated proteins were subject to western blotting with anti-FHL3, anti-YY1, and anti-EZH2 (indicated at the left). IgG was used as a negative control. The total cell lysates before immunoprecipitation were used as input to verify expression of FHL3, EZH2, and YY1. D The GST pulldown results indicated that addition of FHL3 fusion protein reduced the interaction His-EZH2 (465–519) with YY1 in vitro. In vitro translated proteins of GST, GST-YY1, His-EZH2 (465–519), and FHL3 were used for GST pulldown assays. The FHL3 fusion protein, GST, GST-YY1, and His-EZH2 (465–519) were detected by western blotting with anti-FHL3, anti-GST, and anti-His, respectively. E, F ChIP-qPCR results showed the EZH2 (E) and H3K27me3 (F) enrichments at the MyHC2b regulatory regions were significantly decreased after FHL3 overexpression in C2C12 myoblasts differentiated for 2 days. IgG was used as negative control, and the precipitated DNA was amplified by PCR with primers for the MyHC2b regulatory regions. G, H ChIP-qPCR results showed the EZH2 (G) and H3K27me3 (H) enrichments at the MyHC2b regulatory regions were significantly increased after FHL3 knockdown in C2C12 myoblasts differentiated for 2 days. IgG was used as negative control, and the precipitated DNA was amplified by PCR with primers for the MyHC2b regulatory regions. The data were presented as mean ± SD of three independent experiments; **P < 0.01, ***P < 0.001

Article Snippet: Mouse FHL3 small interfering RNA (siRNA) oligonucleotides (sense: CCAUGAGCGAGGCAUUUGATT; anti-sense: UCAAAUGCCUCGCUCAUGGTT); mouse YY1 siRNA oligonucleotides (sense: GGACCUUUACUGCCACAAATT; anti-sense: UUUGUGGCAGUAAAGGUCCTT) and pig FHL3 siRNA Oligonucleotides (siRNA1-sense:AGCGCAAAUACAUUCAGACGTT; siRNA1-anti-sense:CGUCUGAAUGUAUUUGCGCTT; siRNA2-sense:GCUCUGUAACGACUGCUACTT; siRNA2-anti-sense:GUAGCAGUCGUUACAGAGCTT; siRNA3-sense:CCGGGACGAUGAUCCUUAUTT; siRNA3-anti-sense: AUAAGGAUCAUCGUCCCGGTT) were designed and synthesized by GenePharma (China, Shanghai).

Techniques: In Vitro, Western Blot, Co-Immunoprecipitation Assay, Over Expression, Knockdown, In Vivo, Immunoprecipitation, Negative Control, Expressing, ChIP-qPCR, Amplification

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism

doi: 10.1007/s00018-022-04680-w

Figure Lengend Snippet: FHL3 reduces glucose tolerance and its expression is positively correlated with blood glucose values in patients with type 2 diabetes. A Heatmap of DEGs associated with glycolysis, TCA cycle, and OXPHOS. B qRT-PCR results showed that the expression levels of glycolysis-related genes were significantly decreased, and the expression levels of TCA cycle and OXPHOS-related genes were significantly increased in FHL3 knockout cells. C KEGG pathway of DEGs between WT and FHL3 KO C2C12 cells. D GTT results of WT and TG male mice showed the clearance of glucose from the circulation during GTT was significantly lower in TG mice compared with WT mice (n = 8 for each group). E ITT results of WT and TG male mice showed the clearance of glucose from the circulation during ITT was significantly lower in TG mice compared with WT mice (n = 8 for each group). F Volcano map of DEGs between normal people and diabetic patients (GSE: 29221) showed that the mRNA expression of FHL3 in muscle was significantly upregulated in diabetic patients. Blue: downregulated DEGs, red: upregulated DEGs. G Correlation analysis of gene expression data in diabetic patients (GSE: 202295) showed that the mRNA expression of FHL3 in muscle was positively correlated with HbA1c. H Schematic diagram depicting the molecular mechanism of fast glycolytic MyHC2b gene expression regulated by the interaction of FHL3 and YY1. YY1 functional domains contain transactivation domain (YY1-TD; 1–99), repression domain (YY1-RD; 170–225) and DNA-binding domain (YY1-DD; 294–414). YY1-RD domain includes HAT-HDAC interaction domain (YY1-RD1;170–200) and REPO domain (YY1-RD2; 200–225); YY1-DD domain includes four zinc finger domains (ZN): ZN1 (294–322), ZN2 (323–350), ZN3 (351–380) and ZN4 (381–414). FHL3 directly interacts with YY1-ZN2, weakening their binding to the MyHC2b regulatory region and thus upregulating MyHC2b gene expression. Moreover, FHL3 competes with EZH2 to bind YY1-RD2, and decreases H3K27me3 enrichment at the MyHC2b regulatory region, thereby increasing target gene expression. The relative mRNA levels were normalized to β-actin. The data were presented as mean ± SD of three independent experiments; *P < 0.05, **P < 0.01

Article Snippet: Mouse FHL3 small interfering RNA (siRNA) oligonucleotides (sense: CCAUGAGCGAGGCAUUUGATT; anti-sense: UCAAAUGCCUCGCUCAUGGTT); mouse YY1 siRNA oligonucleotides (sense: GGACCUUUACUGCCACAAATT; anti-sense: UUUGUGGCAGUAAAGGUCCTT) and pig FHL3 siRNA Oligonucleotides (siRNA1-sense:AGCGCAAAUACAUUCAGACGTT; siRNA1-anti-sense:CGUCUGAAUGUAUUUGCGCTT; siRNA2-sense:GCUCUGUAACGACUGCUACTT; siRNA2-anti-sense:GUAGCAGUCGUUACAGAGCTT; siRNA3-sense:CCGGGACGAUGAUCCUUAUTT; siRNA3-anti-sense: AUAAGGAUCAUCGUCCCGGTT) were designed and synthesized by GenePharma (China, Shanghai).

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Gene Expression, Functional Assay, Binding Assay, Targeted Gene Expression