Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 1. CBP80 and eIF4E are bound to mRNA in both nuclear and cytoplasmic cell fractions. (A) Structure of the pmCMV-Gl test plasmids, which harbor either a nonsense-free Gl allele that terminates translation at codon 147 (Norm) or a Gl allele carrying a TAG nonsense codon at position 39 (Ter). (B) Cos cells were transiently transfected with a test pmCMV-Gl plasmid and the reference plasmid phCMV-MUP. Nuclear (N) and cytoplasmic (C) fractions were then immunopurified under conditions that preserved RNP using either normal rabbit serum (NRS), anti-CBP80 antibody (α-CBP80) or anti-eIF4E antibody (α-eIF4E), and subjected to western blotting using anti-CBP80 antibody or anti-eIF4E antibody. Nuclear fractions were free of cytoplasm as evidenced by the absence of detectable reactivity with anti-eIF4A antibody (data not shown). For easy visualization, bands corresponding to antibody are not shown. (C) The levels of Gl and MUP mRNAs were quantitated in RNA prepared from each immunopurification using RT–PCR. The leftmost five lanes, which analyze decreasing amounts of RNA before immunopurification (– IP), demonstrate that the conditions of RT–PCR were quantitative. Numbers below the figure represent the level of Gl mRNA normalized to the level of MUP mRNA, where normalized levels of nonsense-free (Norm) mRNA in each anti-CBP80 and anti-eIF4E immunopurification of each fraction are defined as 100%. Results are representative of two independently performed experiments where the efficiency of NMD varied by no more than 12%. The immunopurification using anti-eIF4E and cytoplasmic sample was less efficient for the transfection using pmCMV-Gl Norm than for the transfection using pmCMV-Gl Ter, explaining the reduced levels of eIF4E detected by western blotting (B) and RNA detected by RT–PCR (C).

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immu-Puri, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 2. CBP80 but not eIF4E is detectably bound to intron-containing pre-mRNA. (A) As in Figure 1, except that only nuclear fractions and Gl pre-mRNA rather than Gl mRNA were analyzed. Furthermore, mouse anti-eIF4E antibody was used instead of rabbit anti-eIF4E antibody and mouse IgG served to control for non-specific immunopurification using mouse anti-eIF4E antibody. (B) As in (A) except that rabbit anti-eIF4E antibody was used in place of mouse anti-eIF4E antibody and the analysis of MUP mRNA was omitted. (C) As in (A) except that SV40 pre-mRNA and mRNA were analyzed. Results are representative of other independently performed experiments, including those using rabbit anti-eIF4E antibody and NRS as a control. Taking the sum of Gl or SV40 pre-mRNA immunopurified by anti-CBP80 antibody and anti-eIF4E antibody in each panel as 100%, the amount of CBP80-bound pre-mRNA was 96 ± 6% and the amount of eIF4E-bound pre-mRNA was 4 ± 6%.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 3. CBP80 but neither eIF4E nor Upf2 immunopurifies with the CTD. The nuclear fraction of 32 × 106 untransfected Cos cells was immunopurified using either NRS or antibody to the C-terminal domain of RNA polymerase II (α-CTD), and protein from either 8 × 105 cells (CTD) or 32 × 105 cells (CBP80, eIF4E and Upf2) was subjected to western blotting using anti-CTD, anti-CBP80, anti-eIF4E or anti-Upf2 antibody. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 1.6 × 105 cells (CTD and CBP80) or 6.4 × 105 cells (eIF4E and Upf2) before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. The two forms of CTD probably differ in the degree of phosphorylation (Dubois et al., 1994). Results are representative of 2–4 independently performed experiments, depending on the antibody used in western blotting.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 5. RNPS1, Y14, SRm160, REF/Aly and Upf2 co-immunopurify with Upf3X in an RNase-insensitive manner, while RNPS1, Y14, REF/Aly, TAP, Upf3X and Upf2 co-immunopurify with CBP80 in an RNase-sensitive manner. As in Figure 4 except that an additional immunopurification using anti-Upf3/3X was performed, total (T) lysate was immunopurified using anti-CBP80 antibody, RNase was added to half of each sample prior to immunopurification, RNA as well as protein were prepared, and western blot analyses of eIF4E and DEK were omitted. (A) RT–PCR quantitation of the level of β-actin mRNA before or after immunopurification using anti-Upf3/3X antibody in order to demonstrate the efficiency of RNase treatment. Prior to immununopurification, half of the sample was treated with RNase (+), and the other half was not treated with RNase (–). (B) Western blot analysis of protein immunopurified using anti-Upf3/3X antibody from nuclear and cytoplasmic fractions. (C) Western blot analysis of protein immunopurified using anti-CBP80 antibody from total cell extract.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 4. Nuclear and cytoplasmic CBP80, unlike nuclear and cytoplasmic eIF4E, co-immunopurify with RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2. Nuclear and cytoplasmic fractions of 32 × 106 untransfected Cos cells were immunopurified using anti-CBP80 antibody, anti-eIF4E antibody or, as a control, NRS. Immunopurified protein from 8 × 105 or 32 × 105 cells was then analyzed by western blotting using the antibodies specified. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 8 × 105 or 32 × 105 cells before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. Notably, Upf3 probably co-migrates with the heavy chain of anti-hUpf3/3X antibody and, therefore, was not assayable.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 6. Model for the dynamics of mRNP structure in mammalian cells as a consequence of pre-mRNA capping, pre-mRNA splicing and mRNA export. In the nucleus, CBP80-bound pre-mRNA is spliced to generate CBP80-bound mRNA. This mRNA is bound by an EJC (dark gray shapes), consisting of RNPS1, Y14, SRm160 and REF/Aly, located 20–24 nucleotides upstream of the exon–exon junction. The EJC recruits the NMD factor Upf3/3X and the mRNA export factor TAP. Some mRNPs (possibly those subject to nucleus-associated NMD) undergo remodeling while associated with the nucleus (nucleus-associated remodeling). Other mRNPs (including those subject to cytoplasmic NMD) undergo remodeling after export to the cytoplasm (cytoplasmic remodeling). In both types of remodeling, CBP80 is replaced by eIF4E and the EJC is lost. Data indicate that the mostly perinuclear Upf2 protein is recruited by the EJC at a point when mRNA co-purifies with nuclei. In theory, recruitment could occur during mRNA export. However, if nucleus-associated remodeling takes place in the nucleoplasm, it is conceivable that an as yet undetected pool of Upf2 is nucleoplasmic. The single exon–exon junction exemplified is likely to typify all splicing-generated exon–exon junctions, regardless of position relative to the mRNA 3′ end.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Generated

Journal:

Article Title: Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development

doi: 10.1128/MCB.24.15.6539-6549.2004

Figure Lengend Snippet: Phosphorylation of eIF4E by Mnk1 and Mnk2 in mouse embryonic fibroblasts. (A) Fibroblasts prepared from WT or DKO embryos were serum starved for 20 h and either left unstimulated (−) or stimulated with FCS (25%, 15 min), TPA (500 nM, 15 min), anisomycin (10 μg/ml, 15 min), UV-C irradiation (40 J/m2, then incubated for 20 min), TNF-α (10 ng/ml, 30 min), IL-1β (10 ng/ml, 15 min), or osmotic shock (additional 0.2 M NaCl, 30 min) followed by isotonic recovery (30 min). Cells growing exponentially in DMEM containing 10% FCS were prepared in parallel (growing). Cell lysates (10 μg protein) were analyzed by immunoblotting with anti-phospho-Mnk1 (Thr197/202), anti-phospho-eIF4E (Ser209), anti-eIF4E, anti-phospho-ERK, anti-phospho-p38, and anti-phospho-HSP27/25 antibodies, as indicated on to the right of the panel. The positions of the phospho-Mnk1 and -Mnk2 proteins are indicated by filled and open arrowheads, respectively. (B) Embryonic fibroblasts of the indicated genotypes were treated as described for panel A, and cell lysates (10 μg of protein) were analyzed by immunoblotting with anti-phospho-eIF4E (Ser209), anti-eIF4E, anti-Mnk1, and anti-Mnk2 antibodies. The asterisks indicate nonspecific signals (ns). (C) Embryonic fibroblasts from WT, Mnk1 KO (1-KO), Mnk2 KO (2-KO), or DKO mice were radiolabeled with [32P]orthophosphate under serum-starved conditions for 20 h and then stimulated with 500 nM TPA for 15 min. eIF4E was purified from cell extracts by either m7GTP-Sepharose affinity resin or immunoprecipitation with an anti-eIF4E monoclonal antibody. Samples were resolved by SDS-PAGE, electrotransferred to a PVDF membrane, and exposed to an X-ray film for autoradiography (upper panel). The filter was then subjected to immunoblotting with the anti-eIF4E antibody (lower panel). IgL, immunoglobulin light chain; circled P, phospho.

Article Snippet: For the immunopurification of eIF4E, 1 μg of an anti-eIF4E monoclonal antibody (clone P-2; Santa Cruz) and 20 μl of protein G-Sepharose were added to 0.4 ml of the cell extract and incubated for 1 h at 4°C.

Techniques: Irradiation, Incubation, Western Blot, Purification, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development

doi: 10.1128/MCB.24.15.6539-6549.2004

Figure Lengend Snippet: Phosphorylation of eIF4E by Mnk1 and Mnk2 in mouse tissues. (A) Tissue extracts (18 μg of protein) prepared from WT, Mnk1 KO (1K), Mnk2 KO (2K), and DKO (DK) mice were analyzed by immunoblotting with anti-Mnk1, anti-Mnk2, anti-phospho-eIF4E (Ser209), and anti-eIF4E antibodies. eIF4E was also purified from liver extracts with m7GTP-Sepharose and analyzed similarly by immunoblotting (left side, bottom panels). The asterisks indicate nonspecific signals (ns). (B) LPS-stimulated activation of Mnk1 and upregulation of eIF4E phosphorylation. Mnk1-Mnk2 KO mice (8 weeks old) received an intraperitoneal injection of LPS (100 μg/g of body weight, from E. coli O55:B5; Sigma). Sixty minutes after injection, the spleen and liver were dissected, and their extracts were analyzed by immunoblotting with the indicated antibodies. (C) Insulin-stimulated activation of Mnk1 and upregulation of eIF4E phosphorylation. Following an overnight fast, Mnk1-Mnk2 KO mice (8 weeks old) were injected intravenously with human insulin (100 mU/g of body weight, Novolin R; Novo Nordisk). Fifteen minutes after injection, the femoral muscle and liver were dissected, and their extracts were analyzed by immunoblotting with the indicated antibodies. Circled P, phospho.

Article Snippet: For the immunopurification of eIF4E, 1 μg of an anti-eIF4E monoclonal antibody (clone P-2; Santa Cruz) and 20 μl of protein G-Sepharose were added to 0.4 ml of the cell extract and incubated for 1 h at 4°C.

Techniques: Western Blot, Purification, Activation Assay, Injection

Journal: eLife

Article Title: PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner

doi: 10.7554/eLife.83345

Figure Lengend Snippet: ( A ) qPCR results of overexpression of Ebf1 , Myod , Myog , Gata1, and NFκB1 in MC3T3-E1 cell line. GATA1 and NFκB1 overexpression could significantly upregulate the expression level of Pth1r . Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. ( B ) Representative confocal images of GATA1 and NFκB1 immunofluorescence in Zfp467 +/+ and Zfp467 -/- bone marrow stromal cells (BMSCs) and related quantification. ( C ) Nuclear protein level of GATA1 and NFκB1 in Zfp467 +/+ and Zfp467 -/- BMSCs. ( D ) Quantification analysis for nuclear protein level of GATA1 and NFκB1 in Zfp467 +/+ and Zfp467 -/- BMSCs. Data shown as mean ± SD, n=3 independent experiments for each group. Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. Figure 4—source data 1. Western blot for . Figure 4—source data 2. Western blot for ACTIN. Figure 4—source data 3. Western blot for H3. Figure 4—source data 4. Western blot for p50 in cytoplasm. Figure 4—source data 5. Western blot for GATA1 in cytoplasm. Figure 4—source data 6. Western blot for GATA1 in nuclear. Figure 4—source data 7. Western blot for p50 in nuclear.

Article Snippet: NFκB1 (IB) , Cell Signaling Technology , 13681S.

Techniques: Over Expression, Expressing, Immunofluorescence, Western Blot

Journal: eLife

Article Title: PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner

doi: 10.7554/eLife.83345

Figure Lengend Snippet: ( A ) Reporter assays using the indicated P1 or P2 reporter construct and an expression vector bearing Gata1 , Nfkb1, or a control empty vector. Data shown as mean ± SD by one-way ANOVA, n=3 independent experiments for each group. Data shown as mean ± SD by one-way ANOVA, n=3 independent experiments for each group. ( B ) Immunoblot assay using a control rabbit IgG antibody (IgG) or the anti-NFκB1 antibody during chromatin immunoprecipitation assay. ( C ) DNA enrichment of Pth1r P2 promoter, ratio between NFκB1 and IgG IP products, first part and last two parts of P2 were significantly enriched by NFκB1 antibody. Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. ( D, E ) qPCR results of the expression levels of Nfkb1 and Pth1r in Nfkb1 siRNA-treated Zfp46 +/+ and Zfp467 -/- calvarial osteoblasts (COBs) and bone marrow stromal cells (BMSCs). Data shown as mean ± SD by two-way ANOVA, n=3 independent experiments for each group. ( F ) Western blot analysis of Nfkb1 and Pth1r in Nfkb1 siRNA-treated Zfp467 +/+ and Zfp467 -/- COBs and BMSCs. ( G ) Quantification for PTH1R protein level. Data shown as mean ± SD by two-way ANOVA, n=3 independent experiments for each group. NC, negative control. Figure 5—source data 1. Western blot for . Figure 5—source data 2. Western blot for p50 with IP samples. Figure 5—source data 3. Western blot for PTH1R. Figure 5—source data 4. Western blot for p50. Figure 5—source data 5. Western blot for ACTIN.

Article Snippet: NFκB1 (IB) , Cell Signaling Technology , 13681S.

Techniques: Construct, Expressing, Plasmid Preparation, Control, Western Blot, Chromatin Immunoprecipitation, Negative Control

Journal: eLife

Article Title: PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner

doi: 10.7554/eLife.83345

Figure Lengend Snippet: ( A ) DNA enrichment of Pth1r P2 promoter, ratio between IP and input, first two parts of P2 were enriched using NFκB1 antibody. Data shown as mean ± SD by unpaired Student’s t test, n=3 replicates for each group. ( B ) RT-PCR product of chromatin immunoprecipitation assay using the nuclear extracts from MC3T3-E1 cells. Figure 5—figure supplement 1—source data 1. Nucleic acid blot for . Figure 5—figure supplement 1—source data 2. Nucleic acid blot for , PCR blot for Pth1r amplification product.

Article Snippet: NFκB1 (IB) , Cell Signaling Technology , 13681S.

Techniques: Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification

Journal: eLife

Article Title: PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner

doi: 10.7554/eLife.83345

Figure Lengend Snippet: ( A ) DNA pulldown assay with biotin-labeled Pth1r P2. MC3T3-E1 nuclear extracts or NFκB1 recombinant protein was probed with biotin-Pth1rP2 and then subjected to immunoblotting using NFκB1 antibody. ( B ) qPCR results of the expression levels of Nfyc , Npas1, Rel , Akap8 , Rela , Ankrd42 , Relb, and Hdac1 in related siRNA-treated MC3T3-E1 cells. Data shown as mean ± SD by one-way ANOVA, n=3 independent experiments for each group. NC, negative control. ( C ) qPCR results of the expression levels of Pth1r in Nfkb1 overexpression plasmid and Nfyc , Npas1, Rel , Akap8 , Rela , Ankrd42 , Relb , or Hdac1 siRNA co-transfected MC3T3-E1 cells. NC, negative control. ( D ) IP results using NFκB1 antibody in MC3T3-E1 protein extracts, IgG was used as a negative control. ( E ) Protein level of p-p105 and p50 in Zfp467 knockdown MC3T3-E1 cells. Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. ( F ) Protein and mRNA level of NFκB-inducing kinase (NIK) in control and Zfp467 siRNA-treated MC3T3-E1 cells. Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. ( G ) Protein level of p-IKKa and p-p100 and mRNA level of Ikka in control and Zfp467 siRNA-treated MC3T3-E1 cells. Data shown as mean ± SD by unpaired Student’s t test, n=3 independent experiments for each group. Figure 6—source data 1. Western blot for . Figure 6—source data 2. Western blot for p50 in DNA pulldown experiment using nuclear extract. Figure 6—source data 3. Western blot for p50 in DNA pulldown experiment using p50 purified protein. Figure 6—source data 4. Western blot for RelB. Figure 6—source data 5. Western blot for NPAS1. Figure 6—source data 6. Western blot for p50. Figure 6—source data 7. Western blot for p-p105. Figure 6—source data 8. Western blot for p50/p105. Figure 6—source data 9. Western blot for ACTIN. Figure 6—source data 10. Western blot for NFκB-inducing kinase (NIK). Figure 6—source data 11. Western blot for ACTIN. Figure 6—source data 12. Western blot for p-IKKα/β. Figure 6—source data 13. Western blot for IKKα. Figure 6—source data 14. Western blot for p-p100. Figure 6—source data 15. Western blot for p100.

Article Snippet: NFκB1 (IB) , Cell Signaling Technology , 13681S.

Techniques: Labeling, Recombinant, Western Blot, Expressing, Negative Control, Over Expression, Plasmid Preparation, Transfection, Knockdown, Control, Purification

Journal: eLife

Article Title: PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner

doi: 10.7554/eLife.83345

Figure Lengend Snippet: Antibody list for co-IP, ChIP, and western blot.

Article Snippet: NFκB1 (IB) , Cell Signaling Technology , 13681S.

Techniques: Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

doi: 10.1136/jitc-2020-001481

Figure Lengend Snippet: Figure 3 Evaluation of antitumor efficacy and immune response in vivo after combined-treatment with FIC-PDT and ripasudil. (A) Treatment schedule of FIC-PDT and ripasudil monotherapy and combination therapy. (B) Tumor growth in B16F10 tumor- bearing mice after various treatments (n=9–12 per group). (C) Flow cytometry analyses of the percentage of costimulatory molecules CD40 and CD86 in CD11c+ cells from TDLNs (n=3 per group). (D, E) Tumor-infiltrating CD8+ T cells in tumor site analyzed by flow cytometry (D) or under fluorescent microscope (E) (n=3–6 per group). (F) T cells from TDLNs of B16F10 tumor- bearing mice were stimulated with irradiated B16F10 tumor lysates and IFN-γ production was examined by ELISA. (n=3–4 per group). One-way ANOVA followed by Tukey’s posthoc test was used for statistical analysis; *p<0.05, **p<0.01, ***p<0.001. Data are presented as the mean±SEM. ANOVA, analysis of variance; FIC-PDT, PDT with Ce6-embedded nanophotosensitizer; PDT, photodynamic therapy; TDLN, tumor draining lymph node.

Article Snippet: Prepared slides were stained with primary antibodies for Ki-67 (ab16667, Abcam), F4/80 (NB600-404, Novus), PD- L1, CD4, CD8 (25 229S, 98 941S, 13 684S, Cell Signaling Technology), FOXP3 (14-5773- 82, Invitrogen), for 30 min and then each protein was detected using HRP- polymer conjugated secondary antibodies (ab214880, Abcam; MP-7444, Vector Laboratories).

Techniques: In Vivo, Flow Cytometry, Microscopy, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Journal for immunotherapy of cancer

Article Title: In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

doi: 10.1136/jitc-2020-001481

Figure Lengend Snippet: Figure 4 In vivo PD-L1 expression and combined therapy with FIC-PDT, ripasudil, and anti-PD-L1 antibody. (A) PD-L1 expression of CD11b+ cells in TME assessed by flow cytometry (n=3 per group) (B) Treatment schedule of FIC-PDT, ripasudil and α-PD-L1 antibody. (C) Tumor growth curves (n=8–10 per group) and (D) survival curves (n=5–7 per group) in B16F10 tumor- bearing mice after various treatments. (E) Tumor-infiltrating CD8+ T cells in tumor under fluorescent microscope (n=4 per group). One-way ANOVA followed by Tukey’s posthoc test was used for statistical analysis; *p<0.05, **p<0.01, ***p<0.001. Data are presented as the mean±SEM. Survival was determined using the log‐rank test. ANOVA, analysis of variance; FIC-PDT, PDT with Ce6-embedded nanophotosensitizer; PDT, photodynamic therapy; TME, tumor microenvironment.

Article Snippet: Prepared slides were stained with primary antibodies for Ki-67 (ab16667, Abcam), F4/80 (NB600-404, Novus), PD- L1, CD4, CD8 (25 229S, 98 941S, 13 684S, Cell Signaling Technology), FOXP3 (14-5773- 82, Invitrogen), for 30 min and then each protein was detected using HRP- polymer conjugated secondary antibodies (ab214880, Abcam; MP-7444, Vector Laboratories).

Techniques: In Vivo, Expressing, Flow Cytometry, Microscopy

Journal: Journal for immunotherapy of cancer

Article Title: In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

doi: 10.1136/jitc-2020-001481

Figure Lengend Snippet: Figure 5 Abscopal effect of local administration of FIC-PDT and ripasudil combined with α-PD-L1 antibody. (A) Treatment schedule for abscopal animal model after various treatments. (B) Tumor growth curves of primary and secondary tumors (n=8– 12 per group). (C,D) Percentage of CD103-expressing DCs (C) and CD44+CD8+ T cells (D) in TDLNs. (E,F) Percentage of CD8+ T cells (E) and granzyme B+ T cells (F) in primary and secondary tumors was quantified by flow cytometry. (G) Ratio of CD8+ T cells over regulatory (CD25+CD4+) T cells in primary and secondary tumors. One-way ANOVA followed by Tukey’s posthoc test was used for statistical analysis; *p<0.05, **p<0.01, ***p<0.001. Data are presented as the mean±SEM. ANOVA, analysis of variance; FIC-PDT, PDT with Ce6-embedded nanophotosensitizer; PDT, photodynamic therapy; TDLN, tumor draining lymph node.

Article Snippet: Prepared slides were stained with primary antibodies for Ki-67 (ab16667, Abcam), F4/80 (NB600-404, Novus), PD- L1, CD4, CD8 (25 229S, 98 941S, 13 684S, Cell Signaling Technology), FOXP3 (14-5773- 82, Invitrogen), for 30 min and then each protein was detected using HRP- polymer conjugated secondary antibodies (ab214880, Abcam; MP-7444, Vector Laboratories).

Techniques: Animal Model, Expressing, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

doi: 10.1136/jitc-2020-001481

Figure Lengend Snippet: Figure 7 Population and location of immune cell subsets in tumor using multiplex IHC. (A) Exact location and density of CD8+ cells (green) and PD-L1+ cells (red) in the TME were investigated using multiplex IHC. (B) Tumor tissues were stained with Ki67 (green), PD-L1 (red), FOXP3 (orange), CD8 (white) and nuclei was costained with DAPI (blue). Yellow arrows represents the proliferative CD8+ T cells. (C) Percentage of proliferative CD8+ (Ki67+CD8+) cells. IHC, immunohistochemistry; TME, tumor microenvironment.

Article Snippet: Prepared slides were stained with primary antibodies for Ki-67 (ab16667, Abcam), F4/80 (NB600-404, Novus), PD- L1, CD4, CD8 (25 229S, 98 941S, 13 684S, Cell Signaling Technology), FOXP3 (14-5773- 82, Invitrogen), for 30 min and then each protein was detected using HRP- polymer conjugated secondary antibodies (ab214880, Abcam; MP-7444, Vector Laboratories).

Techniques: Multiplex Assay, Staining, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeted inhibition of Nrf2 potentiates antitumor immunity and enhances the efficacy of immunotherapy in hepatocellular carcinoma

doi: 10.1136/jitc-2024-010841

Figure Lengend Snippet: Nrf2 inhibition enhances antitumor immunity by upregulating immune response genes, increasing immune cell infiltration, and boosting CTLs activity. ( A ) Volcano plot depicting the fold changes of genes of BRU-treated tumor tissues versus control ones. Dots in red represent upregulated genes (log2(FC) ≥1 and adjusted p value≤0.05) and dots in blue represent downregulated genes (log2(FC) ≤−1 and adjusted p value p ≤ 0.05) in BRU-treated tumor tissues versus control ones. Highlighted genes are involved in innate and adaptive immune response pathways. Statistical analysis was performed using Wald test with Benjamini-Hochberg correction. ( B ) GOBP analysis of highly expressed genes in BRU-treated tumor tissues versus control ones. N=4 biologically independent samples per group. ( C ) Gene set enrichment analysis for immune-related pathway in BRU-treated tumor tissues versus control ones. N=4 biologically independent samples per group (adjusted p value<0.05 and |NES|>1). ( D ) Representative immunohistochemical staining of Nrf2, PD-L1. Scale bar, 100 µm. ( E ) Immunofluorescence analysis of active cleaved caspase-3 in tumor tissues. Scale bar, 50 µm. ( F ) Immunofluorescence analysis of CD8 and granzyme B in tumor tissues. Scale bar, 50 µm. For ( D ), ( E ) and ( F ), data represent mean±SEM, n=5. ( G–J ) Flow cytometry analysis: CD8 + T cells infiltrating per gram tumor mass ( G ); ( H ) Left: representative flow cytometry plots depicting the expression of granzyme B, IFN-γ, and CD107a. Right: statistical analysis of the relative MFI of GzmB per CD8 + T cells (GzmB expression was assessed by measuring the MFI of APC-GzmB within the CD8 + T-cell population. Data are presented as “Relative MFI”, calculated as the ratio of the MFI of individual samples to the mean MFI of the control group within the same experiment, N=4; statistical analysis of the IFN-γ + , and CD107a + cells among the CD8 + T-cell population, N=5; MDSC, DC, Macrophages, FoxP3 + Treg, and NK cell numbers infiltrating per gram tumor mass ( I ); percentages of M1 and M2 type macrophages ( J ); N=4. Statistical significance in G and H was determined using unpaired t-tests; statistical significance in I and J was determined by one-way ANOVA. Data represent the mean±SEM. *p<0.05. analysis of variance; BRU, brusatol; CTL, cytotoxic T lymphocyte; DC, dendritic cell; FC, fold change; GOBP, Gene Ontology Biological Process; GzmB, granzyme B; IFN, interferon; MFI, mean fluorescence intensity; MDSC, myeloid-derived suppressor cell; NES, normalized enrichment score; NK, natural killer; Nrf2, nuclear factor erythroid 2-related factor 2; n.s., not significant; PD-L1, programmed death ligand-1; Treg, regulatory T cell.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: Nrf2 (#12721, 1:1000 for WB, 1:200 for IHC, 1:200 for IF), Anti-rat IgG, Alexa Fluor 488 (#4416S, 1:500 for IF), PD-L1 (#13684, 1:1000 for WB; #64988, 1:200 for IF, 1:50 for IHC), NF-κB p65 (#8242, 1:1000), Phospho-NF-κB p65 (#3033, 1:1000), Cleaved Caspase-3 (#9664, 1:200); from Beyotime: Nrf2 (#AF7623, 1:1000 for WB), PD-L1 (#AF7710, 1:1000 for WB); from Proteintech: Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (#SA00009-2, 1:200), GAPDH (#10494-1-AP, 1:8000); from AiFang Biology: CD8 (#AFRM0004, 1:200 for IF), Granzyme B (#AFRM0352, 1:200 for IF); from BioLegend: anti-mouse CD45 (#103137, #103138, #103154), anti-mouse CD3ε (#100306, #100216), anti-mouse CD4 (#100451), anti-mouse CD8α (#100722, #100792), anti-human/mouse Granzyme B (#372204, #396408, Isotype ctrl #400141), anti-mouse IFN-γ (#505830, Isotype ctrl #400429), anti-mouse CD107a (#121612, Isotype ctrl #400507), anti-mouse CD274 (#124308, 1:100), anti-mouse CD25 (#102063), anti-mouse F4/80 (#123162), anti-mouse/human CD11b (#101257), anti-mouse CD86 (#105032), anti-mouse Gr-1 (#108416), anti-mouse CD11c (#117328) and anti-human HLA-A2 (#343307); from BD Biosciences (anti-mouse): CD206 (#568273), FoxP3 (#562996).

Techniques: Inhibition, Activity Assay, Control, Immunohistochemical staining, Staining, Immunofluorescence, Flow Cytometry, Expressing, Fluorescence, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeted inhibition of Nrf2 potentiates antitumor immunity and enhances the efficacy of immunotherapy in hepatocellular carcinoma

doi: 10.1136/jitc-2024-010841

Figure Lengend Snippet: Nrf2 inhibition attenuates PD-L1 expression at the transcriptional level in cancer cells. ( A ) Cell surface PD-L1 expression was analyzed after 24 hours for the indicated treatment (IFN-γ, 100 ng/mL) by flow cytometry. ( B ) Time-course western blot analysis of Nrf2 and PD-L1 protein levels in Hepa1-6 cells stimulated with IFN-γ (100 ng/mL) in the absence or presence of BRU (40 nM) treatment. Cells were harvested at the indicated time points (24 hours and 48 hours). Western blot analysis of PD-L1 expression in Huh7 stable KD Nrf2 cells ( C ) and Huh7 cells with ectopic overexpression of Nrf2 ( D ). ( E ) RT-PCR analysis of Hepa1-6 cells treated with BRU (40 nM) or along with IFN-γ (100 ng/mL). ( F ) and ( G ) Impact of BRU treatment on PD-L1 levels at the transcription level was measured via dual-luciferase reporter assay. Error bars denote mean±SEM. ****p<0.0001; ***p<0.001; **p<0.01. BRU, brusatol; IFN, interferon; KD, knockdown; MFI, mean fluorescence intensity; mRNA, messenger RNA; Nrf2, nuclear factor erythroid 2-related factor 2; n.s., not significant; PD-L1, programmed death ligand-1; RT-PCR, real-time PCR.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: Nrf2 (#12721, 1:1000 for WB, 1:200 for IHC, 1:200 for IF), Anti-rat IgG, Alexa Fluor 488 (#4416S, 1:500 for IF), PD-L1 (#13684, 1:1000 for WB; #64988, 1:200 for IF, 1:50 for IHC), NF-κB p65 (#8242, 1:1000), Phospho-NF-κB p65 (#3033, 1:1000), Cleaved Caspase-3 (#9664, 1:200); from Beyotime: Nrf2 (#AF7623, 1:1000 for WB), PD-L1 (#AF7710, 1:1000 for WB); from Proteintech: Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (#SA00009-2, 1:200), GAPDH (#10494-1-AP, 1:8000); from AiFang Biology: CD8 (#AFRM0004, 1:200 for IF), Granzyme B (#AFRM0352, 1:200 for IF); from BioLegend: anti-mouse CD45 (#103137, #103138, #103154), anti-mouse CD3ε (#100306, #100216), anti-mouse CD4 (#100451), anti-mouse CD8α (#100722, #100792), anti-human/mouse Granzyme B (#372204, #396408, Isotype ctrl #400141), anti-mouse IFN-γ (#505830, Isotype ctrl #400429), anti-mouse CD107a (#121612, Isotype ctrl #400507), anti-mouse CD274 (#124308, 1:100), anti-mouse CD25 (#102063), anti-mouse F4/80 (#123162), anti-mouse/human CD11b (#101257), anti-mouse CD86 (#105032), anti-mouse Gr-1 (#108416), anti-mouse CD11c (#117328) and anti-human HLA-A2 (#343307); from BD Biosciences (anti-mouse): CD206 (#568273), FoxP3 (#562996).

Techniques: Inhibition, Expressing, Flow Cytometry, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Knockdown, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeted inhibition of Nrf2 potentiates antitumor immunity and enhances the efficacy of immunotherapy in hepatocellular carcinoma

doi: 10.1136/jitc-2024-010841

Figure Lengend Snippet: Nrf2 inhibition potentiates the efficacy of PD-1 antibody against HCC in vivo. C57BL/6J mice bearing allograft HCC tumors (implanted with 1×10 7 Hepa1-6 cells) received BRU or anti-PD-1 antibody treatment or their combination. n=5 mice. ( A ) Study design. ( B ) Photographs of C57BL/6J mice tumors isolated on day 20. ( C ) Endpoint tumor weight. N=5 ( D ) Tumor growth curves for each group. Comparison of the tumor size after different treatments at day 20. Data represent the mean±SEM. Statistical significance was assessed using two-way ANOVA, **p<0.01; *p<0.05. ( E ) CD3 + , CD4 + , CD8 + T cells infiltrating per gram tumor mass, n=4. ( F–G ) Flow cytometry analysis of cell-surface PD-L1 ( F ), MHC-I ( G ) in tumor cells. n=4 mice. Statistical significance in E, F and G was determined by one-way ANOVA. Data represent the mean±SD. ****p<0.0001; **p<0.01; *p<0.05. ( H ) and ( I ) Immunofluorescent staining of active CCA3, CD8, and granzyme B in the tumors, and the quantification of active CCA3, CD8 and granzyme B signals. Scale bars, 50 µm. n=5 different areas of the indicated tissues for each mouse. Statistical significance in C, H and I was determined by one-way ANOVA. Data represent the mean±SEM. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ANOVA, analysis of variance; BRU, brusatol; CCA, cleaved caspase-3; HCC, hepatocellular carcinoma; i.p., intraperitoneal; MFI, mean fluorescence intensity; MHC, major histocompatibility complex class I; NES, normalized enrichment score; Nrf2, nuclear factor erythroid 2-related factor 2; n.s., not significant; PD-1, programmed death-1; PD-L1, programmed death ligand-1.

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: Nrf2 (#12721, 1:1000 for WB, 1:200 for IHC, 1:200 for IF), Anti-rat IgG, Alexa Fluor 488 (#4416S, 1:500 for IF), PD-L1 (#13684, 1:1000 for WB; #64988, 1:200 for IF, 1:50 for IHC), NF-κB p65 (#8242, 1:1000), Phospho-NF-κB p65 (#3033, 1:1000), Cleaved Caspase-3 (#9664, 1:200); from Beyotime: Nrf2 (#AF7623, 1:1000 for WB), PD-L1 (#AF7710, 1:1000 for WB); from Proteintech: Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (#SA00009-2, 1:200), GAPDH (#10494-1-AP, 1:8000); from AiFang Biology: CD8 (#AFRM0004, 1:200 for IF), Granzyme B (#AFRM0352, 1:200 for IF); from BioLegend: anti-mouse CD45 (#103137, #103138, #103154), anti-mouse CD3ε (#100306, #100216), anti-mouse CD4 (#100451), anti-mouse CD8α (#100722, #100792), anti-human/mouse Granzyme B (#372204, #396408, Isotype ctrl #400141), anti-mouse IFN-γ (#505830, Isotype ctrl #400429), anti-mouse CD107a (#121612, Isotype ctrl #400507), anti-mouse CD274 (#124308, 1:100), anti-mouse CD25 (#102063), anti-mouse F4/80 (#123162), anti-mouse/human CD11b (#101257), anti-mouse CD86 (#105032), anti-mouse Gr-1 (#108416), anti-mouse CD11c (#117328) and anti-human HLA-A2 (#343307); from BD Biosciences (anti-mouse): CD206 (#568273), FoxP3 (#562996).

Techniques: Inhibition, In Vivo, Isolation, Comparison, Flow Cytometry, Staining, Fluorescence, Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeted inhibition of Nrf2 potentiates antitumor immunity and enhances the efficacy of immunotherapy in hepatocellular carcinoma

doi: 10.1136/jitc-2024-010841

Figure Lengend Snippet: Working model for Nrf2 inhibition by Brusatol promotes antitumor immunity in HCC. Nrf2 inhibition downregulates PD-L1 expression at the transcriptional level and enhances MHC-I expression via NF-κB activation, leading to the activation of CD8 + T cell, granzyme B secretion thereby killing cancer cells (This figure is drawn by Figdraw). HCC, hepatocellular carcinoma; MHC, major histocompatibility complex class I; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor erythroid 2-related factor 2; PD-1, programmed death-1; PD-L1, programmed death ligand-1; TCR, T-cell receptor. All abbreviations used in this study are listed in .

Article Snippet: Primary antibodies against the following proteins were obtained from Cell Signaling Technology: Nrf2 (#12721, 1:1000 for WB, 1:200 for IHC, 1:200 for IF), Anti-rat IgG, Alexa Fluor 488 (#4416S, 1:500 for IF), PD-L1 (#13684, 1:1000 for WB; #64988, 1:200 for IF, 1:50 for IHC), NF-κB p65 (#8242, 1:1000), Phospho-NF-κB p65 (#3033, 1:1000), Cleaved Caspase-3 (#9664, 1:200); from Beyotime: Nrf2 (#AF7623, 1:1000 for WB), PD-L1 (#AF7710, 1:1000 for WB); from Proteintech: Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (#SA00009-2, 1:200), GAPDH (#10494-1-AP, 1:8000); from AiFang Biology: CD8 (#AFRM0004, 1:200 for IF), Granzyme B (#AFRM0352, 1:200 for IF); from BioLegend: anti-mouse CD45 (#103137, #103138, #103154), anti-mouse CD3ε (#100306, #100216), anti-mouse CD4 (#100451), anti-mouse CD8α (#100722, #100792), anti-human/mouse Granzyme B (#372204, #396408, Isotype ctrl #400141), anti-mouse IFN-γ (#505830, Isotype ctrl #400429), anti-mouse CD107a (#121612, Isotype ctrl #400507), anti-mouse CD274 (#124308, 1:100), anti-mouse CD25 (#102063), anti-mouse F4/80 (#123162), anti-mouse/human CD11b (#101257), anti-mouse CD86 (#105032), anti-mouse Gr-1 (#108416), anti-mouse CD11c (#117328) and anti-human HLA-A2 (#343307); from BD Biosciences (anti-mouse): CD206 (#568273), FoxP3 (#562996).

Techniques: Inhibition, Expressing, Activation Assay, Immunopeptidomics

Journal: Cancer cell international

Article Title: MTMR6 downregulation contributes to cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s12935-025-03654-9

Figure Lengend Snippet: Fig. 3 IHC analysis of OSCC tissues from patients for MTMR6 protein expression. A Tissues from patients with OSCC showing cisplatin-sensitive and cisplatin-insensitive tissues were stained with an anti-MTMR6 antibody (Tissue S1-S6:cisplatin-sensitive, Tissue R1-R6: cisplatin-insensitive). The protein expression level on each specimen was scored as 0, 1, 2, and 3 (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) according to its staining intensity (*p < 0.05). B Kaplan–Meier survival plots for MTMR6 expression were generated with the TCGA dataset of HNSCC patients (n = 515). C Kaplan–Meier survival plots for MTMR6 were generated with the TCGA dataset of HNSCC patients with cisplatin-treatment history (n = 96)

Article Snippet: Antibodies against MTMR6 and KRT13, developed in rabbits, were supplied by Proteintech (Rosemont, IL, USA), while mouse anti-β-actin (HRP-conjugated) antibodies were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Generated

Journal: Cancer cell international

Article Title: MTMR6 downregulation contributes to cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s12935-025-03654-9

Figure Lengend Snippet: Fig. 4 hsa-miR-544a downregulated MTMR6 expression. A miR-544a mRNA expression level was compared in UMSCC1 and UM-Cis cells by RT-qPCR. B Predicted miR-544a binding sites in 3′-UTR of MTMR6 mRNA are shown. Mutations in the MTMR6 3′-UTR are shown in red. Luciferase reporter constructs were generated with the wild-type (WT) and mutant (MT) 3′-UTRs of MTMR6. C Dual luciferase reporter activity demonstrates the target relationship between miR-544a and MTMR6 mRNA. The activity was measured in UM-Cis cells transfected with the WT and MT 3′-UTR MTMR6 luciferase constructs. The activity was normalized to that of Renilla luciferase. D UMSCC1 cells were transfected with miR-544a mimic for 24 h, and cisplatin was treated for another 2 days. MTMR6 mRNA and protein expression were analyzed using RT-qPCR and Western blot analysis. E Cell viability was measured under the same conditions using an MTT assay. F Apoptosis was measured under the same experimental condition using Western blot analysis. G UM-Cis cells were transfected with anti-miR-544a for 24 h, and cisplatin was treated for another 2 days. The effect of anti-miR544a on MTMR6 mRNA and protein expression was analyzed using RT-qPCR and Western blot analysis. H Cisplatin efficacy was evaluated under the same conditions via MTT assay. Results represent the mean ± standard deviation of three experiments (*p < 0.05, **p < 0.01)

Article Snippet: Antibodies against MTMR6 and KRT13, developed in rabbits, were supplied by Proteintech (Rosemont, IL, USA), while mouse anti-β-actin (HRP-conjugated) antibodies were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Construct, Generated, Mutagenesis, Activity Assay, Transfection, Western Blot, MTT Assay, Standard Deviation

Journal: Cancer cell international

Article Title: MTMR6 downregulation contributes to cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s12935-025-03654-9

Figure Lengend Snippet: Fig. 5 Effect of MTMR6 knockdown on cisplatin sensitivity in UMSCC1-derived mouse xenografts. A UMSCC1 spheroids were formed in 96-well U-bottom ultra-low attachment plates and transfected with siMTMR6 for 24 h. The spheroids were then subcutaneously injected into mice on the right and left side of the backs of six mice. (100 per tumor, approximately 1 × 106 cells). After tumor formation, the mice were divided into cisplatin injection (n = 3) and vehicle control (n = 3) in each siControl and siMTMR6 group. Cisplatin (2.5 mg/kg) or DMSO (0.1% v/v in PBS) vehicle control was intraperitoneally injected twice a week and sacrificed on the 18th day after cisplatin administration. Tumor volume was measured using a caliper till sacrifice. Results represent the mean ± standard deviation (*p < 0.05; **p < 0.005). B IHC staining for KRT13 and MTMR6 in mice xenograft tissues. H&E staining images are presented as controls. C MTMR6-positive cells were counted in the immunostained mice tumor tissues (***p < 0.001). D MTMR6 mRNA expression was measured in mice tumor tissues using RT-qPCR analysis. The fold-change of each mRNA is represented as the mean ± standard deviation (**p < 0.005)

Article Snippet: Antibodies against MTMR6 and KRT13, developed in rabbits, were supplied by Proteintech (Rosemont, IL, USA), while mouse anti-β-actin (HRP-conjugated) antibodies were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Derivative Assay, Transfection, Injection, Control, Standard Deviation, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR