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Novus Biologicals anti ing4
Anti Ing4, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 2 article reviews

anti ing4 - by Bioz Stars, 2026-05

93/100 stars

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Figure 1. Loss of ING4 promoted tumor immune escape. A), The correlation of ING4 expression with T cell inﬁltration was assayed using TIME 2. http://timer.cistrome.org/. B), The correlation of ING4 expression with activated CD8+ T cell abundance was assayed using TISIDB (hku.hk). C), Im- munohistochemical analysis of ING4, PD-L1, and CD8 using LUSC tumor tissue specimens. Scale bar: 100 μm. The correlation of ING4 with PD-L1, and CD8 protein expression was assayed (n = 48). D), Western blot analysis of wild type (WT) or ING4−/- H520 cell lysates. The relative PD-L1 level

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

doi: 10.1002/advs.202304068

Figure Lengend Snippet: Figure 1. Loss of ING4 promoted tumor immune escape. A), The correlation of ING4 expression with T cell inﬁltration was assayed using TIME 2. http://timer.cistrome.org/. B), The correlation of ING4 expression with activated CD8+ T cell abundance was assayed using TISIDB (hku.hk). C), Im- munohistochemical analysis of ING4, PD-L1, and CD8 using LUSC tumor tissue specimens. Scale bar: 100 μm. The correlation of ING4 with PD-L1, and CD8 protein expression was assayed (n = 48). D), Western blot analysis of wild type (WT) or ING4−/- H520 cell lysates. The relative PD-L1 level

Article Snippet: Antibodies, Immunoprecipitation, and Western Blot: ING4 (10617-1- AP), CK2α (10992-1-AP), Tubulin (11224-1-AP), Lamp1(21997-1-AP), PDL1(66248-1), ATG7 (10088-2-AP), LC3B (14600-1-AP), GST (66001-2), and Flag (66008-4) antibodies were purchased from Proteintech.

Techniques: Expressing, Western Blot

Figure 2. ING4 induced PD-L1 autophagic degradation. A), H520 cells were transfected vector (pcDNA3) or Flag-ING4 plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-ING4/∆NLS plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. C, H520 cells were transfected vector (pcDNA3) or Flag-ING4 plasmids for 48 h. Cells were treated with cycloheximide (CHX, 30 μg ml−1) as indicated time course. Cell lysates were subjected to Western blot analysis. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. D, WT or ING4−/- H520 cells were treated with CHX (30 μg ml−1) as indicated time course. Cell lysates were subjected to Western blot analysis. The relative PD-L1 level was quantiﬁed. Results are expressed as means ±

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

doi: 10.1002/advs.202304068

Figure Lengend Snippet: Figure 2. ING4 induced PD-L1 autophagic degradation. A), H520 cells were transfected vector (pcDNA3) or Flag-ING4 plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-ING4/∆NLS plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. C, H520 cells were transfected vector (pcDNA3) or Flag-ING4 plasmids for 48 h. Cells were treated with cycloheximide (CHX, 30 μg ml−1) as indicated time course. Cell lysates were subjected to Western blot analysis. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. D, WT or ING4−/- H520 cells were treated with CHX (30 μg ml−1) as indicated time course. Cell lysates were subjected to Western blot analysis. The relative PD-L1 level was quantiﬁed. Results are expressed as means ±

Techniques: Transfection, Plasmid Preparation, Western Blot

Figure 3. The interaction of ING4 with PD-L1. A), Immunoprecipitation and Western blot analysis of H520 cell lysates. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B), The interaction of ING4 with PD-L1 was assayed by confocal in H520 cells. Scale bar: 25 μm. C), Schematic illustration of PD-L1. D), H520 cells were transfected vector (pcDNA3), his-PD-L1 or his-PD-L1/∆C plasmids as indicated for 48 h. Cell lysates were subjected to Ni-NTA pull-down and Western blot. The relative binding of his-PD-L1 to ING4 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. E), Schematic illustration of ING4. PHD: plant homeodomain. F), H520 cells were transfected PEBG (GST), GST-ING4, or GST-ING4/∆PHD plasmids as indicated. Cell lysates were subjected to GST pull-down and Western blot. The relative binding of GST-ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

doi: 10.1002/advs.202304068

Figure Lengend Snippet: Figure 3. The interaction of ING4 with PD-L1. A), Immunoprecipitation and Western blot analysis of H520 cell lysates. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B), The interaction of ING4 with PD-L1 was assayed by confocal in H520 cells. Scale bar: 25 μm. C), Schematic illustration of PD-L1. D), H520 cells were transfected vector (pcDNA3), his-PD-L1 or his-PD-L1/∆C plasmids as indicated for 48 h. Cell lysates were subjected to Ni-NTA pull-down and Western blot. The relative binding of his-PD-L1 to ING4 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. E), Schematic illustration of ING4. PHD: plant homeodomain. F), H520 cells were transfected PEBG (GST), GST-ING4, or GST-ING4/∆PHD plasmids as indicated. Cell lysates were subjected to GST pull-down and Western blot. The relative binding of GST-ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Transfection, Plasmid Preparation

Figure 4. LIR motif of ING4 was required for PD-L1 autophagic degradation and anti-tumor immune escape. A), Immunoprecipitation and Western blot analysis of H520 cell lysates. The relative binding of ING4 to LC3B was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. B), Alignment of ING4 with LIR motif contained proteins, and schematic illustration of ING4 LIR motif. C), in vitro binding analysis of the interaction of ING4 with LC3B as described in methods. Down panel was ponceau S staining. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. D, H520 cells were transfected PEBG (GST), GST-ING4, GST-F178A plasmids as indicated for 48 h. GST pull-down analysis was performed using cell lysates. The relative binding of ING4 to LC3B was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. E, WT or ATG7−/- H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. F, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 cells, and co-localization of PD-L1 with LAMP1 was detected by confocal. Scale bar: 25 μm. Percent colocalization of PD-L1 with LAMP1 was quantiﬁed. Results are expressed as means ± SEM (n = 15 ﬁelds). *P<0.05. G,H, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 h and co-cultured with Jurkat cells for 24 h. IL-2 or IFN-𝛾release from Jurkat cells was assayed. Results are expressed as means ± SEM (n = 3). *P<0.05. I,J), Implanted tumor model analysis of vector (pLenti-CMV), ING4 or F178A stably expressing LLC cells. Tumor volume and weight were measured. Results are expressed as means ± SEM, n = 6. *P<0.05.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

doi: 10.1002/advs.202304068

Figure Lengend Snippet: Figure 4. LIR motif of ING4 was required for PD-L1 autophagic degradation and anti-tumor immune escape. A), Immunoprecipitation and Western blot analysis of H520 cell lysates. The relative binding of ING4 to LC3B was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. B), Alignment of ING4 with LIR motif contained proteins, and schematic illustration of ING4 LIR motif. C), in vitro binding analysis of the interaction of ING4 with LC3B as described in methods. Down panel was ponceau S staining. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. D, H520 cells were transfected PEBG (GST), GST-ING4, GST-F178A plasmids as indicated for 48 h. GST pull-down analysis was performed using cell lysates. The relative binding of ING4 to LC3B was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. E, WT or ATG7−/- H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 h and Western blot analysis of cell lysates. The relative PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.01. F, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 cells, and co-localization of PD-L1 with LAMP1 was detected by confocal. Scale bar: 25 μm. Percent colocalization of PD-L1 with LAMP1 was quantiﬁed. Results are expressed as means ± SEM (n = 15 ﬁelds). *P<0.05. G,H, H520 cells were transfected vector (pcDNA3), Flag-ING4 or Flag-F178A plasmids for 48 h and co-cultured with Jurkat cells for 24 h. IL-2 or IFN-𝛾release from Jurkat cells was assayed. Results are expressed as means ± SEM (n = 3). *P<0.05. I,J), Implanted tumor model analysis of vector (pLenti-CMV), ING4 or F178A stably expressing LLC cells. Tumor volume and weight were measured. Results are expressed as means ± SEM, n = 6. *P<0.05.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, In Vitro, Staining, Transfection, Plasmid Preparation, Cell Culture, Stable Transfection, Expressing

Figure 8. Inhibitor of CK2 enhanced antitumor immunotherapy. A), H520 cells were treated without or with TBB (10 μM), TBB (10 μM)+ CQ (30 μM), or TBB (10 μM)+ MG132 (20 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B), WT or ATG7−/- H520 cells were treated without or with TBB (10 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. C), H520 cells were treated with TBB (10 μM) for 0, 0.5, and 2 h. Cell lysates were subjected to immunoprecipitation and Western blot. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. D), WT or ING4−/- H520 cells were treated without or with TBB (10 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. E,F), co-cultured Jurkat cells with H520 cells were treated with TBB (10 μM) for 24 h. IL-2 or IFN-𝛾production from Jurkat cells was detected. Results are expressed as means ± SEM (n = 3). *P<0.05. G,H), LLC cells were inoculated subcutaneously C57BL/6 mice. Mice were treated control, TBB, anti-PD-1 mice monoclonal antibody, or TBB+anti-PD-1 antibody as described in method. Tumor volume and weight were detected. Results are expressed as means±SEM, n = 5. *P<0.05. I), Relative surface PD-L1 expression in LLC cell implanted tumors was assayed by ﬂow cytometry. MFI: median ﬂuorescence intensity. Results are expressed as means ± SEM (n = 5). *P<0.05. J), The percentage of CD8+ /IFN-𝛾+ T cells in LLC cell implanted tumors was assayed by ﬂow cytometry. Results are expressed as means ± SEM (n = 5). *P<0.05.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CK2/ING4 Pathway Facilitates Non-Small Cell Lung Cancer Immunotherapy.

doi: 10.1002/advs.202304068

Figure Lengend Snippet: Figure 8. Inhibitor of CK2 enhanced antitumor immunotherapy. A), H520 cells were treated without or with TBB (10 μM), TBB (10 μM)+ CQ (30 μM), or TBB (10 μM)+ MG132 (20 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. B), WT or ATG7−/- H520 cells were treated without or with TBB (10 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. C), H520 cells were treated with TBB (10 μM) for 0, 0.5, and 2 h. Cell lysates were subjected to immunoprecipitation and Western blot. The relative binding of ING4 to PD-L1 was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. D), WT or ING4−/- H520 cells were treated without or with TBB (10 μM) for 4 h and Western blot analysis of cell lysates. PD-L1 level was quantiﬁed. Results are expressed as means ± SEM (n = 3). *P<0.05. E,F), co-cultured Jurkat cells with H520 cells were treated with TBB (10 μM) for 24 h. IL-2 or IFN-𝛾production from Jurkat cells was detected. Results are expressed as means ± SEM (n = 3). *P<0.05. G,H), LLC cells were inoculated subcutaneously C57BL/6 mice. Mice were treated control, TBB, anti-PD-1 mice monoclonal antibody, or TBB+anti-PD-1 antibody as described in method. Tumor volume and weight were detected. Results are expressed as means±SEM, n = 5. *P<0.05. I), Relative surface PD-L1 expression in LLC cell implanted tumors was assayed by ﬂow cytometry. MFI: median ﬂuorescence intensity. Results are expressed as means ± SEM (n = 5). *P<0.05. J), The percentage of CD8+ /IFN-𝛾+ T cells in LLC cell implanted tumors was assayed by ﬂow cytometry. Results are expressed as means ± SEM (n = 5). *P<0.05.

Techniques: Western Blot, Immunoprecipitation, Binding Assay, Cell Culture, Control, Expressing, Cytometry

ING4 promoted the formation of RCC cell sphere. (A–B) Lentivirus-mediated ING4 and its vector (Vec) was stably overexpressed in the Ketr-3 and 786-O cells and relative protein expression of ING4 was normalized to that of the respective α-tubulin. (C–D) Photographs and number of cell spheres in ING4 overexpression (ING4) and control (Vec) Ketr-3 and 786-O cells (n = 3). (E–F) Three lentivirus-mediated ING4-targeted sgRNAs (sgRNA-ING4) and vector (sgRNA-Vec) were used to stably knockdown the ING4 expression in Ketr-3 and 786-O cells and relative protein expression of ING4 was normalized to that of the respective α-tubulin. (G–H) Photographs and number of cell spheres in ING4 knockdown and control Ketr-3 and 786-O cells (n = 3). Image magnification, ×100; scale bar, 100 μm; data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4 promoted the formation of RCC cell sphere. (A–B) Lentivirus-mediated ING4 and its vector (Vec) was stably overexpressed in the Ketr-3 and 786-O cells and relative protein expression of ING4 was normalized to that of the respective α-tubulin. (C–D) Photographs and number of cell spheres in ING4 overexpression (ING4) and control (Vec) Ketr-3 and 786-O cells (n = 3). (E–F) Three lentivirus-mediated ING4-targeted sgRNAs (sgRNA-ING4) and vector (sgRNA-Vec) were used to stably knockdown the ING4 expression in Ketr-3 and 786-O cells and relative protein expression of ING4 was normalized to that of the respective α-tubulin. (G–H) Photographs and number of cell spheres in ING4 knockdown and control Ketr-3 and 786-O cells (n = 3). Image magnification, ×100; scale bar, 100 μm; data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001.

Article Snippet: The sections were blocked in 3% BSA for 30 min, and then incubated with polyclonal rabbit anti-CD44 antibody (GB14037, 1:300 dilution; Servicebio, China), anti-MYC antibody (GB13076, 1:300 dilution; Servicebio, China), anti-NANOG antibody (GB11331, 1:500 dilution; Servicebio, China), and anti-ING4 (HPA057338, 1:5,000 dilution; Sigma-Aldrich, United States) at 4°C overnight and washed with PBS (pH 7.4) three times for 5 min each.

Techniques: Plasmid Preparation, Stable Transfection, Expressing, Over Expression, Standard Deviation

ING4 increased the expression of stem cell markers in RCC cells. (A–B) Real-time PCR was used to test the relative mRNA expression of stem cell markers OCT4, CD44, NANOG, and MYC in ING4-overexpressed or knockdown Ketr-3 and 786-O cells compared to the respective vector control. (C–E) Western blot was performed to test the protein expression levels of OCT4, CD44, NANOG, and MYC in ING4-overexpressed or knockdown Ketr-3 and 786-O cells and relative protein expression was normalized to that of the respective α-tubulin. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4 increased the expression of stem cell markers in RCC cells. (A–B) Real-time PCR was used to test the relative mRNA expression of stem cell markers OCT4, CD44, NANOG, and MYC in ING4-overexpressed or knockdown Ketr-3 and 786-O cells compared to the respective vector control. (C–E) Western blot was performed to test the protein expression levels of OCT4, CD44, NANOG, and MYC in ING4-overexpressed or knockdown Ketr-3 and 786-O cells and relative protein expression was normalized to that of the respective α-tubulin. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Standard Deviation

ING4-promoted tumor growth and lung metastasis in vivo . (A–B) The picture of xenograft tumors and the size change of these tumors arising from ING4-overexpressed (ING4) and vector control (Vec) 786-O cells from 30 to 42 days. (C) The representative lung images of mice and HE staining of the lung in the stable ING4-overexpressed group (ING4) and control 786-O group (Vec). (D) The number of lung metastatic nodules was counted. (E–F) The representative IHC images and IRS staining scores of ING4, MYC, CD44, and NANOG in the primary tumors collected from the tumor-bearing experiment and metastatic lesions collected from the tail vein metastasis model (n = 5). IHC image magnification, ×200; scale bar, 100μm; data were presented as mean ± standard deviation, * p < 0.05, ** p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4-promoted tumor growth and lung metastasis in vivo . (A–B) The picture of xenograft tumors and the size change of these tumors arising from ING4-overexpressed (ING4) and vector control (Vec) 786-O cells from 30 to 42 days. (C) The representative lung images of mice and HE staining of the lung in the stable ING4-overexpressed group (ING4) and control 786-O group (Vec). (D) The number of lung metastatic nodules was counted. (E–F) The representative IHC images and IRS staining scores of ING4, MYC, CD44, and NANOG in the primary tumors collected from the tumor-bearing experiment and metastatic lesions collected from the tail vein metastasis model (n = 5). IHC image magnification, ×200; scale bar, 100μm; data were presented as mean ± standard deviation, * p < 0.05, ** p < 0.001.

Techniques: In Vivo, Plasmid Preparation, Staining, Standard Deviation

ING4-increased ISG expression. (A) The volcano plot was used to indicate the significantly differential gene expressions in 786-O cells. (B) GO analysis was used to identify the functional enrichments in the biological process, the vertical coordinate was the enriched GO term, and the horizontal coordinate was the number of significantly different genes in that term. (C–D) The real-time PCR analyzed the mRNA expression levels of ISGs, including OAS2, MX2, IFITM1, and IFITM2 in ING4-overexpressed (ING4) 786-O and Ketr-3 cells compared to the respective vector controls (Vec). (E–F) The relative mRNA expression levels of ISGs in ING4 knockdown (sgRNA-ING4) Ketr-3 and 786-O cells were compared to the respective vector control (sgRNA-Vec). Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4-increased ISG expression. (A) The volcano plot was used to indicate the significantly differential gene expressions in 786-O cells. (B) GO analysis was used to identify the functional enrichments in the biological process, the vertical coordinate was the enriched GO term, and the horizontal coordinate was the number of significantly different genes in that term. (C–D) The real-time PCR analyzed the mRNA expression levels of ISGs, including OAS2, MX2, IFITM1, and IFITM2 in ING4-overexpressed (ING4) 786-O and Ketr-3 cells compared to the respective vector controls (Vec). (E–F) The relative mRNA expression levels of ISGs in ING4 knockdown (sgRNA-ING4) Ketr-3 and 786-O cells were compared to the respective vector control (sgRNA-Vec). Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001.

Techniques: Expressing, Functional Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Standard Deviation

ING4 promoted stemness enrichment of RCC cells through the activation of the p38 MAPK/type I IFN-stimulated gene signaling pathway. (A–B) Western blot showed levels of p38 MAPK and Erk1/2 activation in ING4-overexpressed (ING4) or knockdown (sgRNA-ING4) Ketr-3 and 786-O cells, and the relative protein expression of p-p38 or p-Erk1/2 was normalized to that of the respective p38 or Erk1/2. (C–D) Real-time PCR analyzed the mRNA expression levels of ISGs including OAS2, MX2, IFITM1, and IFITM2 in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h. (E–F) Photographs and number of cell spheres in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 pretreatment for 24 h (n = 3). (G–H) Western blot tested the protein expression levels of stem cell markers OCT4, CD44, MYC, and NANOG in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h and relative protein expression was normalized to that of the respective α-tubulin. Note: images magnification, ×100; Scale bar, 100 μm; Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001, ns: no significance.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4 promoted stemness enrichment of RCC cells through the activation of the p38 MAPK/type I IFN-stimulated gene signaling pathway. (A–B) Western blot showed levels of p38 MAPK and Erk1/2 activation in ING4-overexpressed (ING4) or knockdown (sgRNA-ING4) Ketr-3 and 786-O cells, and the relative protein expression of p-p38 or p-Erk1/2 was normalized to that of the respective p38 or Erk1/2. (C–D) Real-time PCR analyzed the mRNA expression levels of ISGs including OAS2, MX2, IFITM1, and IFITM2 in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h. (E–F) Photographs and number of cell spheres in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 pretreatment for 24 h (n = 3). (G–H) Western blot tested the protein expression levels of stem cell markers OCT4, CD44, MYC, and NANOG in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h and relative protein expression was normalized to that of the respective α-tubulin. Note: images magnification, ×100; Scale bar, 100 μm; Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001, ns: no significance.

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Plasmid Preparation, Standard Deviation

ING4 inhibited the DUSP4 expression to regulate the p38 MAPK activity. (A–B) The mRNA correlation of ING4 with DUSP4 in TCGA-KIRC and TCGA-KIRP from the starBase v3.0 project. (C–D) Real-time PCR was used to test the relative mRNA expression levels of DUSP4 in ING4-overexpressed (ING4) or knockdown (sgRNA-ING4) Ketr-3 and 786-O cells compared to the respective vector controls. (E–F) Western blot was used to test the protein expression of DUSP4 in ING4 overexpression (ING4) or knockdown (sgRNA-ING4) or their respective vectors Ketr-3 and 786-O cells and relative protein expression was normalized to that of respective α-tubulin. (G–H) Western blot was used to test the expression of ING4, DUSP4, p-p38, and p38 in ING4 knockdown (sgRNA-ING4-2) or vector control (sgRNA-Vec) Ketr-3 and 786-O cells after transfection with DUSP4 (si-DUSP4) or control siRNA (si-Con), and the relative protein expression of ING4 was normalized to that of the respective α-tubulin. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: ING4 inhibited the DUSP4 expression to regulate the p38 MAPK activity. (A–B) The mRNA correlation of ING4 with DUSP4 in TCGA-KIRC and TCGA-KIRP from the starBase v3.0 project. (C–D) Real-time PCR was used to test the relative mRNA expression levels of DUSP4 in ING4-overexpressed (ING4) or knockdown (sgRNA-ING4) Ketr-3 and 786-O cells compared to the respective vector controls. (E–F) Western blot was used to test the protein expression of DUSP4 in ING4 overexpression (ING4) or knockdown (sgRNA-ING4) or their respective vectors Ketr-3 and 786-O cells and relative protein expression was normalized to that of respective α-tubulin. (G–H) Western blot was used to test the expression of ING4, DUSP4, p-p38, and p38 in ING4 knockdown (sgRNA-ING4-2) or vector control (sgRNA-Vec) Ketr-3 and 786-O cells after transfection with DUSP4 (si-DUSP4) or control siRNA (si-Con), and the relative protein expression of ING4 was normalized to that of the respective α-tubulin. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.001.

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Over Expression, Transfection, Standard Deviation

Inhibition of DUSP4 expression significantly reversed the reduction of cell sphere formation after ING4 knockdown. (A) Photographs of cell spheres in ING4 knockdown (sgRNA-ING4-2) or vector control (sgRNA-Vec) Ketr-3 and 786-O cells after transfection with DUSP4 (si-DUSP4) or control siRNA (si-Con) (n = 3). (B–E) The number of cell spheres with diameters ≥50 μm or ≥100 μm. Image magnification, ×100; scale bar, 100μm; data are presented as mean ± standard deviation. *p < 0.05.

Journal: Frontiers in Pharmacology

Article Title: ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

doi: 10.3389/fphar.2022.845097

Figure Lengend Snippet: Inhibition of DUSP4 expression significantly reversed the reduction of cell sphere formation after ING4 knockdown. (A) Photographs of cell spheres in ING4 knockdown (sgRNA-ING4-2) or vector control (sgRNA-Vec) Ketr-3 and 786-O cells after transfection with DUSP4 (si-DUSP4) or control siRNA (si-Con) (n = 3). (B–E) The number of cell spheres with diameters ≥50 μm or ≥100 μm. Image magnification, ×100; scale bar, 100μm; data are presented as mean ± standard deviation. *p < 0.05.

Techniques: Inhibition, Expressing, Plasmid Preparation, Transfection, Standard Deviation

Confluent PrECs induced to differentiate with 2ng/mL KGF and 5nM R1881 for 3-14 days. ‘L’ indicates only the luminal cells were analyzed. A) Levels of pCREB1, CREB, ING4, and PTEN during differentiation measured by immunoblotting. GAPDH served as loading control. B,C) PrEC-TetON-shCREB1 cells differentiated 5-12 days with (+Dox) or without (-Dox) 25ng/ml doxycycline. B) CREB1, ATF1, and ING4 expression measured by immunoblotting. C) Differentiated cultures immunostained for ING4, nuclei counterstained with Hoescht (Merge), and imaged by phase and fluorescence microscopy. D) Log 2 -fold expression of ING4 mRNA in PrEC-TetON-shCREB1 cells treated with (Dox) or without (Veh) doxycycline measured by qRT-PCR and normalized to day 0. *** p <0.005; ****p<0.001; n=4; error bars SD. E) ChIP of CREB1 at day 3 or 10 of differentiation versus IgG control. Enrichment of CREB1 binding at two CRE elements in the ING4 promoter. F) ChIP of CREB1 at day 3 or 10 of differentiation or day 3 of differentiation with cells overexpressing ING4 (I). G) Immunostaining of PrEC and PrEC-shPTEN for pCREB1.

Journal: Oncogene

Article Title: Aberrant CREB1 Activation in Prostate Cancer Disrupts Normal Prostate Luminal Cell Differentiation

doi: 10.1038/s41388-021-01772-y

Figure Lengend Snippet: Confluent PrECs induced to differentiate with 2ng/mL KGF and 5nM R1881 for 3-14 days. ‘L’ indicates only the luminal cells were analyzed. A) Levels of pCREB1, CREB, ING4, and PTEN during differentiation measured by immunoblotting. GAPDH served as loading control. B,C) PrEC-TetON-shCREB1 cells differentiated 5-12 days with (+Dox) or without (-Dox) 25ng/ml doxycycline. B) CREB1, ATF1, and ING4 expression measured by immunoblotting. C) Differentiated cultures immunostained for ING4, nuclei counterstained with Hoescht (Merge), and imaged by phase and fluorescence microscopy. D) Log 2 -fold expression of ING4 mRNA in PrEC-TetON-shCREB1 cells treated with (Dox) or without (Veh) doxycycline measured by qRT-PCR and normalized to day 0. *** p <0.005; ****p<0.001; n=4; error bars SD. E) ChIP of CREB1 at day 3 or 10 of differentiation versus IgG control. Enrichment of CREB1 binding at two CRE elements in the ING4 promoter. F) ChIP of CREB1 at day 3 or 10 of differentiation or day 3 of differentiation with cells overexpressing ING4 (I). G) Immunostaining of PrEC and PrEC-shPTEN for pCREB1.

Article Snippet: Immunoblotting: ING4 (EP3804) and ATF1 (EPR1590) antibodies were purchased from Abcam. pCREB-Ser 133(87G3), CREB (D76D11) and PTEN (138G6) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Quantitative RT-PCR, Binding Assay, Immunostaining

A) JFK mRNA levels during differentiation measured by qRT-PCR. B) ChIP of CREB1 on the CRE (+CRE) or non-CRE region (−CRE) of the JFK promoter. C) ChIP of ING4 at the transcriptional initiatin site (TIS) of the JFK promoter at day 3 or 10 post differentiation or at day 3 in cells overexpressing ING4 (I). D) Log 2 -fold expression of JFK mRNA in PrEC-TetON-shCREB1 cells treated with (Dox) or without (Veh) doxycycline measured by qRT-PCR and normalized to Day 0. ** p <0.01; *** p <0.001; n=4; error bars SD

Journal: Oncogene

Article Title: Aberrant CREB1 Activation in Prostate Cancer Disrupts Normal Prostate Luminal Cell Differentiation

doi: 10.1038/s41388-021-01772-y

Figure Lengend Snippet: A) JFK mRNA levels during differentiation measured by qRT-PCR. B) ChIP of CREB1 on the CRE (+CRE) or non-CRE region (−CRE) of the JFK promoter. C) ChIP of ING4 at the transcriptional initiatin site (TIS) of the JFK promoter at day 3 or 10 post differentiation or at day 3 in cells overexpressing ING4 (I). D) Log 2 -fold expression of JFK mRNA in PrEC-TetON-shCREB1 cells treated with (Dox) or without (Veh) doxycycline measured by qRT-PCR and normalized to Day 0. ** p <0.01; *** p <0.001; n=4; error bars SD

Techniques: Quantitative RT-PCR, Expressing

A TMA containing 81 tumors and 48 adjacent normal samples was interrogated by IHC for the expression of AR, pCREB1, ING4, and PTEN. A) Levels of expression of each indicated marker in the tumor samples. 0-1 scored as negative, 2-3 scored as positive. B) Fisher test demonstrating the association between low ING4 and low PTEN. ****p<0.0001. C) Representative IHC images.

Journal: Oncogene

Article Title: Aberrant CREB1 Activation in Prostate Cancer Disrupts Normal Prostate Luminal Cell Differentiation

doi: 10.1038/s41388-021-01772-y

Figure Lengend Snippet: A TMA containing 81 tumors and 48 adjacent normal samples was interrogated by IHC for the expression of AR, pCREB1, ING4, and PTEN. A) Levels of expression of each indicated marker in the tumor samples. 0-1 scored as negative, 2-3 scored as positive. B) Fisher test demonstrating the association between low ING4 and low PTEN. ****p<0.0001. C) Representative IHC images.

Techniques: Expressing, Marker

A) Doxycycline concentration required to knock-down CREB1 in EMP-TetON-shCREB1 cells. B) Validation of ERG, MYC, and PTEN expression in tumorigenic EMP cells relative to normal PrEC by immunoblotting. C,D) EMP-TetON-shCREB1 cells differentiated for 12 days with (+Dox) or without (−Dox) 100ng/ml doxycycline. Untreated 12-day differentiated PrECs were included as a control. Cultures were immunostained for C) integrin α6 (ITGα6, basal marker) and AR (luminal marker) or D) cleaved caspase 3 (Casp3) and counterstained with Hoescht (Merge), and imaged by phase or fluorescence microscopy. E) Growth of EMP-TetON-shCREB1 orthotopic tumors in SCID mice treated with (Dox) or without (Suc) doxycycline. * p <0.05; n=6; error bars SD. F) Models for how PTEN influences CREB1 activation and ING4 expression dynamics in normal (PrEC) versus tumor (EMP) cells. G) Pathways by which PTEN and CREB1 control ING4 expression.

Journal: Oncogene

Article Title: Aberrant CREB1 Activation in Prostate Cancer Disrupts Normal Prostate Luminal Cell Differentiation

doi: 10.1038/s41388-021-01772-y

Figure Lengend Snippet: A) Doxycycline concentration required to knock-down CREB1 in EMP-TetON-shCREB1 cells. B) Validation of ERG, MYC, and PTEN expression in tumorigenic EMP cells relative to normal PrEC by immunoblotting. C,D) EMP-TetON-shCREB1 cells differentiated for 12 days with (+Dox) or without (−Dox) 100ng/ml doxycycline. Untreated 12-day differentiated PrECs were included as a control. Cultures were immunostained for C) integrin α6 (ITGα6, basal marker) and AR (luminal marker) or D) cleaved caspase 3 (Casp3) and counterstained with Hoescht (Merge), and imaged by phase or fluorescence microscopy. E) Growth of EMP-TetON-shCREB1 orthotopic tumors in SCID mice treated with (Dox) or without (Suc) doxycycline. * p <0.05; n=6; error bars SD. F) Models for how PTEN influences CREB1 activation and ING4 expression dynamics in normal (PrEC) versus tumor (EMP) cells. G) Pathways by which PTEN and CREB1 control ING4 expression.

Techniques: Concentration Assay, Expressing, Western Blot, Marker, Fluorescence, Microscopy, Activation Assay

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