Journal: Human & experimental toxicology

Article Title: LINC00641 impeded the malignant biological behaviors of papillary thyroid carcinoma cells via interacting with IGF2BP1 to reduce GLI1 mRNA stability.

doi: 10.1177/09603271231180856

Figure Lengend Snippet: Figure 4. LINC00641 physically interacted with IGF2BP1 in PTC cells. (a) The location of LINC00641 was explored through subcellular fractionation. (b) The binding sites of LINC00641 and IGF2BP1 was predicted by starBase. (c–d) RNA pull-down and RIP assays showed association of IGF2BP1 with LINC00641. (e) RT-qPCR was applied to detect the expression of LINC00641. (F) Western blot was applied to detect the expression of IGF2BP1. *p < 0.05.

Article Snippet: The antibodies against IGF2BP1 (#sc166,344, 1:1000), GLI1 (#sc-515,751, 1:1000), E-cadherin (#sc-8426, 1:1000), N-cadherin (#sc-8424, 1: 1000), AKT (#sc-5298, 1:1000), p-AKT (#sc-514,032, 1: 1000), or GAPDH (#sc-365,062, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Fractionation, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Human & experimental toxicology

Article Title: LINC00641 impeded the malignant biological behaviors of papillary thyroid carcinoma cells via interacting with IGF2BP1 to reduce GLI1 mRNA stability.

doi: 10.1177/09603271231180856

Figure Lengend Snippet: Figure 5. LINC00641 destabilized GLI1 mRNA by interacting with IGF2BP1. (a) RIP assay showed association of IGF2BP1 with GLI1. (b) Western blotting was applied to detect the protein expression of GLI1. (c–d) TPC-1 cells were transfected with LINC00641 overexpression plasmid (pcDNA-LINC00641) or shRNA (sh-LINC00641), and RIP assay was applied to analyze the enrichment of GLI1 mRNA in RNA-protein complexes pulled down by anti-IGF2BP1 antibody. (e) Western blotting was applied to detect the protein expression of GLI1. (f–g) The degradation rate of GLI1 mRNA was evaluated by RT-qPCR. *p < 0.05.

Article Snippet: The antibodies against IGF2BP1 (#sc166,344, 1:1000), GLI1 (#sc-515,751, 1:1000), E-cadherin (#sc-8426, 1:1000), N-cadherin (#sc-8424, 1: 1000), AKT (#sc-5298, 1:1000), p-AKT (#sc-514,032, 1: 1000), or GAPDH (#sc-365,062, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, shRNA, Quantitative RT-PCR

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: Interferon-independent restriction of HSV-1 replication in primary murine microglia by ZBP1. Panel ( A ) Microglia derived from ZBP1-expressing (ZBP1+/+) or ZBP1-deficient (ZBP1-/-) mice were infected with HSV-1(MacIntyre) at an MOI of 2 for 1 h. Following infection, cells were either untreated or exposed to 10 µM fludarabine. Supernatants were collected at 24h post-infection and standard plaque assays were performed in Vero cells to determine the number of plaque-forming units (PFU) released from HSV-1(MacIntyre) infected microglia. Panel ( B ) Murine microglia derived from ZBP1-expressing or ZBP1-deficient mice were uninfected (Mock) or infected with HSV-1(MacIntyre) at an MOI of 0.2 or 2. At 24 h post-infection, the concentration of IL-6, IL-18, TNF, and IFN-β, were quantified by ELISA. Data are shown as the mean of 3–6 independent experiments ± SEM. An asterisk indicates a significant difference from similarly treated ZBP1+/+ cells and dagger symbols indicate a significant difference from uninfected cells ( p < 0.05; n = 3)

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Derivative Assay, Expressing, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: HSV-1-induced cell death in primary murine microglia is mediated by ZBP-1. ZBP1-expressing and ZBP1-deficient microglia were infected with a clinical strain of HSV-1 (HSV-1(MacIntyre)) or a laboratory strain (HSV-1(F)). One-hour post-infection, cells were treated with a vehicle control (DMSO), the RIPK1 inhibitor GSK547 (50 nM), the RIPK3 inhibitor GSK843 (2 µM), and/or the caspase 8 inhibitor Z-IETD-FMK (20 µM). Cell viability was measured with a RealTime-Glo™ MT assay every 2 h post-infection starting at hour 2. Data are reported as the final percentage of cell death at 24 h relative to non-infected cells and as the rate of cell death. Data are shown as the mean of 3–5 independent experiments ± SEM. Asterisks indicate a significant difference from similarly treated ZBP1-expressing cells, while dagger symbols indicate significant difference from similarly challenged cells treated with DMSO vehicle only ( p < 0.05)

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Expressing, Infection, Control

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: HSV-1-induced necroptosis occurs in both a RIPK1-independent ZBP1-mediated and a RIPK-mediated ZBP1-independent manner in murine microglia. ZBP1-expressing and ZBP1-deficient microglia were infected with HSV-1(MacIntyre). One-hour post-infection, cells were treated with DMSO as a vehicle control (Panels A and B ) or GSK547 (50 nM) (Panels C and D ). Panels A and C : Cell viability was measured with a RealTime-Glo™ MT assay every 2 h post-infection, starting at hour 2. Panels B and D : 24 h post-infection, whole-cell lysates were collected and analyzed for pMLKL expression or β-actin by immunoblot analysis. Relative pMLKL expression was determined by densitometric analysis and normalized to β-actin expression to correct for changes in total protein loading attributable to cell death. Representative Western blots and the mean densitometric values ± SEM of 3 independent experiments are shown. An asterisk indicates a significant difference in final cell death or pMLKL expression from similarly treated ZBP1-expressing cells and dagger symbols indicate a significant difference from vehicle-treated cells ( p < 0.05)

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Expressing, Infection, Control, Western Blot

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: ZBP1-mediated necroptosis is the primary cell death pathway occurring in HSV-1-infected murine microglia. ZBP1-expressing and ZBP1-deficient microglia were infected with HSV-1(MacIntyre). One-hour post-infection, cells were treated with either GSK843 (2 µM; Panels A and B ) or Z-IETD-FMK (20 µM; Panels C and D ). Panels A and C; Cell viability was measured with a RealTime-Glo™ MT assay every 2 h post-infection, starting at hour 2. Panels B and D ) 24 h post-infection, whole-cell lysates were collected and analyzed for phosphorylated MLKL expression or β-actin by immunoblot analysis. Relative phosphorylated MLKL expression was determined by densitometric analysis and normalized to β-actin expression to correct for changes in total protein loading attributable to cell death. Representative immunoblots and the mean densitometric values ± SEM of 3 independent experiments are shown. An asterisk indicates a significant difference in final cell death or P-MLKL expression from similarly treated ZBP1-expressing cells and dagger symbols indicate a significant difference from vehicle-treated cells ( p < 0.05)

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Infection, Expressing, Western Blot

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: HSV-1-induced necroptosis is mediated by RIPK3 in murine microglia. ZBP1-expressing and ZBP1-deficient microglia were infected with HSV-1(MacIntyre). One-hour post-infection, cells were treated with a combination of GSK547 (50 nM) and Z-IETD-FMK (20 µM) (Panel A ) or a combination of GSK843 (2 µM) and Z-IETD-FMK (20 µM) (Panel B ). Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection. Data are shown as the mean of 4–6 independent experiments ± SEM. Asterisk in Panel A indicates a significant difference in final cell death percentage from similarly treated ZBP1+/+ cells ( p < 0.05)

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Expressing, Infection

Journal: Journal of Neurovirology

Article Title: Z-DNA binding protein 1 mediates necroptotic cell death in primary murine microglia following herpes simplex virus-1 infection

doi: 10.1007/s13365-025-01304-4

Figure Lengend Snippet: Proposed mechanisms for the activation of cell death pathways in murine microglia following challenge with a clinical and a laboratory strain of HSV-1. Infection with HSV-1(MacIntyre) or HSV-1(F) leads to the release of viral DNA and recognition by ZBP1 and its subsequent interaction with RIPK3 mediated by their respective RHIM domains, which results in MLKL phosphorylation and necroptotic cell death. Additionally, HSV-1(MacIntyre) and HSV-1(F) infection causes the production and release of TNF, that can act in an autocrine or paracrine manner to initiate ZBP1-independent necroptosis via RIPK1 activation, in the absence of caspase-8, and its subsequent interaction with RIPK3. In contrast to the clinical strain, TNF activity induced by HSV-1(F) laboratory strain infection may induce apoptosis via caspase-8

Article Snippet: Membranes were blocked with either 5% milk (for ZBP1) or 5% BSA (for pMLKL) for 1 h and then incubated overnight at 4 °C with primary antibodies directed against ZBP1 (AdipoGen Cat# AG-20B-0010-C100) or pMLKL (Abcam Cat# ab187091) or β-actin (Abcam Cat# ab8227).

Techniques: Activation Assay, Infection, Phospho-proteomics, Activity Assay

Journal: Cell Death & Disease

Article Title: ZBP1-mediated PANoptosis is a crucial lethal form in diverse keratinocyte death modalities in UVB-induced skin injury

doi: 10.1038/s41419-025-07351-3

Figure Lengend Snippet: Primer sequences in this study.

Article Snippet: The primary antibody against ZBP1 (#13285-1-AP for human) was from Proteintech (Rosemont, IL, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: ZBP1-mediated PANoptosis is a crucial lethal form in diverse keratinocyte death modalities in UVB-induced skin injury

doi: 10.1038/s41419-025-07351-3

Figure Lengend Snippet: A Constitutive proteins related to PANoptosome including ZBP1, AIM2, NLRP3, NLRP12, RIPK1, RIPK3, p-RIPK3, caspase-8, cleaved caspase-1, FADD, and ASC in mice epidermal lysates were detected by western blotting assay at 48 h after 430 mJ/cm 2 UVB irradiation. B Proteins of N-GSDMD, cleaved PARP, cleaved caspase-3, p-MLKL, and ZBP1 in human primary keratinocytes were detected by western blotting assay after UVB irradiation. C Representative images of immunohistochemical staining of ZBP1 in human skin samples from sun-exposed and non-exposed areas. Statistical analysis of average optical density of ZBP1 positive staining was shown (n = 12). D Representative photos of WT mice and Zbp1 −/− mice at 48 h after 430 mJ/cm 2 UVB exposure. E UVB skin damage of WT mice and Zbp1 −/− mice was evaluated by scores (n = 5). F Representative images of H&E staining and TUNEL staining of skin sections from WT mice and Zbp1 −/− mice after UVB irradiation. The statistic of epidermal thickness and average optical density of TUNEL positive staining were analyzed (n = 5). G Representative images of MPO, MMP9, and F4/80 immunohistochemical staining in WT mice and Zbp1 −/− mice after UVB irradiation. The statistic of average optical densities of MPO, MMP9, and F4/80 positive staining were analyzed (n = 5). H Proteins of ZBP1, N-GSDMD, cleaved PARP, cleaved caspase-3, and p-MLKL in the epidermis of WT mice and Zbp1 −/− mice were detected by western blotting assay. The relative protein levels of the above proteins were compared with ACTB as quantification (n = 3). The scale bar in black is 100 μm for 10× magnification in C , F , and G . The scale bar in yellow is 20 μm for 40× magnification in C and F . *P < 0.05. **P < 0.01. ns not significant. N-GSDMD N-terminal GSDMD. p-MLKL phosphorylated MLKL.

Article Snippet: The primary antibody against ZBP1 (#13285-1-AP for human) was from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Irradiation, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: Expression pattern of IGF2BP1, IGF2BP2, and IGF2BP3 mRNAs in fetal and adult human tissues. IGF2BP1, IGF2BP2, and IGF2BP3 mRNA expression in (A) human fetal liver and adult bone marrow samples and (B) cultured cord blood and adult blood erythroblasts. Mean value ± SD is shown. AB, adult blood erythroblasts; BM, adult bone marrow; CB, cord blood erythroblasts; FL, fetal liver.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Expressing, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1 overexpression has cytoplasmic distribution and robust effects upon hemoglobin expression patterns in adult CD34(+) cells. (A) IGF2BP1 protein expression in Human Fetal Liver (FL) Protein Medley, adult bone marrow (BM), cord blood (CB), and adult blood (AB) erythroblasts. Beta-actin was used as a loading control. Molecular weight is shown in kilodaltons (kDa). IGF2BP1 overexpression (OE) was confirmed by (B) RT-qPCR, (C) Western blot, and (D–I) confocal analyses. RT-qPCR was performed at culture day 14. Mean value ± SD of three independent donors for each condition is shown. Western blot analysis confirmation of IGF2BP1 overexpression at culture day 14 in CD34(+) cells versus empty vector control (C) from three healthy adult donors (3 μg per lane). Beta-actin was used as a loading control. Molecular weight is shown in kilodaltons. Confocal analysis was performed at culture day 12 in IGF2BP1 overexpression cells versus empty vector control. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and IGF2BP1 (green). HPLC analyses of hemoglobin from (J) empty vector control and (K) IGF2BP1 overexpression samples were performed at culture day 21. Datasets are representative of three independent donors for each condition. HbF and HbA peaks are labeled on each graph (y axis, mVolts; x axis, elution time in minutes). P value was calculated using two-tailed Student’s t test. *P < 0.05.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Over Expression, Expressing, Control, Molecular Weight, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Staining, Labeling, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: Quantitative analyses of the percentage of (A) day 14 CD71(+)/GPA(+), (B) day 14 CD71(+)/GPA(−), (C) day 21 CD71(+)/GPA(+), and (D) day 21 CD71(−)/GPA(+) measured by fluorescence-activated cell sorter analysis of control and IGF2BP1 overexpression in adult CD34(+) cells. Mean value ± SD of three independent donors for each condition is shown. P value was calculated using two-tailed Student's t test. *P < 0.05. IGF2BP1-OE increases in fetal hemoglobin are maintained after terminal differentiation. HPLC analyses of hemoglobin at culture day 21 from sorted IGF2BP1-OE (E) enucleated cells and (F) nucleated cells in adult CD34(+) cells. HbF and HbA peaks are labeled on each graph (y axis: mVolts; x axis: elution time in minutes). C, empty vector control; CD71, anti-transferrin receptor; GPA, anti-glycophorin A; OE, IGF2BP1 overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Fluorescence, Control, Over Expression, Two Tailed Test, Labeling, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: Effects of IGF2BP1 overexpression upon differentiation, maturation, enucleation, and cellular morphology of adult CD34(+) cells. Representative flow dot plots at culture day 21 stained for CD71 and GPA from (A) empty vector control cells and (B) IGF2BP1 overexpression. Thiazole orange staining was used to assess enucleation for (C) empty vector control and (D) IGF2BP1 overexpression at culture day 21. Sorted enucleated cells were imaged in (E) empty vector control and (F) IGF2BP1 overexpression after Wright–Giemsa staining. Datasets are representative of three independent donors for each condition. CD71, anti-transferrin receptor; GPA, anti-glycophorin A.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Over Expression, Staining, Plasmid Preparation, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1-OE causes a more fetal-like pattern of gene expression. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for (A) epsilon-globin, (B) gamma-globin, (C) delta-globin, (D) beta-globin, (E) zeta-globin, (F) mu-globin, (G) alpha-globin, (H) theta-globin, (I) CA1, and (J) GCNT2. RT-qPCR analyses were performed at culture day 14. Open bars represent control transductions and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P values were calculated using two-tailed Student’s t test. *P < 0.05. C, empty vector control transduction; OE, IGF2BP1 overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Two Tailed Test, Plasmid Preparation, Transduction, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1-OE has minor effects on selected known IGF2BP1 targets c-MYC, IGF2, and beta-actin. IGF2BP1-OE and control transductions were investigated for the mRNA levels of (A) c-MYC, (B) IGF2, and (C) beta-actin in adult CD34(+) cells. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. (D) Western analyses of c-MYC, IGF2, and beta-actin expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 overexpression in adult CD34(+) cells. Alpha-tubulin was used as a loading control. Molecular weight is shown in kilodaltons (kDa). C, empty vector control; OE, IGF2BP1 overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Control, Plasmid Preparation, Western Blot, Expressing, Over Expression, Molecular Weight

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1-OE has no major effects on the let-7 miRNAs. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for (A) let-7a, (B) let-7b, (C) let-7c, (D) let-7d, (E) let-7e, (F) let-7f, (G) let-7g, (H) let-7i, and (I) miR-98. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P value was calculated using two-tailed Student’s t test. *P < 0.05. C, empty vector control transduction; OE, IGF2BP1 overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Control, Plasmid Preparation, Two Tailed Test, Transduction, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1-OE regulates BCL11A and HMGA2 in human adult erythroblasts. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for the mRNA levels of (A) BCL11A, (B) HMGA2, (C) ZBTB7A, (D) KLF1, and (E) SOX6. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P values were calculated using two-tailed Student’s t test. *P < 0.05. Western blot analyses of (F) BCL11A, HMGA2, and ZBTB7A and (G) KLF1 and SOX6 expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 overexpressed CD34(+) cells are shown. Histone H3 and lamin B1 were used as loading controls. Molecular weight is shown in kilodaltons (kDa). C, empty vector control transduction; OE, IGF2BP1 overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Control, Plasmid Preparation, Two Tailed Test, Western Blot, Expressing, Molecular Weight, Transduction, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1 knockdown in cord blood erythroblasts was confirmed by (A) RT-qPCR and (B) Western blot analyses at culture day 14. Open bar represents empty vector control and black bar represents IGF2BP1 knockdown. Mean value ± SD of three independent donors for each condition is shown. P value was calculated using two-tailed Student’s t test. *P < 0.05. Beta-actin was used as a loading control. Molecular weight is shown in kilodaltons (kDa). C, empty vector control transduction; KD, IGF2BP1 knockdown.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Control, Two Tailed Test, Molecular Weight, Transduction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: Effects upon differentiation, maturation, and enucleation of IGF2BP1 knockdown or IGF2BP1 overexpression compared with each respective control transduction in cord blood CD34(+) cells. Representative flow dot plots at culture (A) day 14 and (B) day 21 stained for CD71 and GPA. (C) Thiazole orange staining was used to assess enucleation at culture day 21. Datasets are representative of three independent donors for each condition. CD71, anti-transferrin receptor; control 1, empty vector control transduction for knockdown vector; control 2, empty vector control transduction for overexpression vector; GPA, anti-glycophorin A; KD, knockdown; OE, overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Knockdown, Over Expression, Control, Transduction, Staining, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: Protein analyses of IGF2BP1 knockdown or overexpression in cord blood erythroblasts. Western images for IGF2BP1, IGF2BP3, LIN28B, BCL11A, HMGA2, and ZBTB7A expression using protein extracts at culture day 14. Lamin B1, histone H3, and beta-actin were used as loading controls. Molecular weight is shown in kilodaltons (kDa). Percentage of fetal hemoglobin (%HbF) shown corresponds to the representative HPLC profiles shown in Fig. S7. Control 1, empty vector control transduction for knockdown vector; control 2, empty vector control transduction for overexpression vector; KD, knockdown; OE, overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Knockdown, Over Expression, Western Blot, Expressing, Molecular Weight, Control, Plasmid Preparation, Transduction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: HPLC analyses of hemoglobin in cord blood CD34(+) cells at culture day 21. (A) Control transduction for knockdown vector (control 1) and control transduction for overexpression vector (control 2). (B) IGF2BP1 knockdown (KD) or IGF2BP1 overexpression (OE). Datasets are representative of triplicate experiments. HbF and HbA peaks are labeled on each graph (y axis, mVolts; x axis, elution time in minutes).

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: Control, Transduction, Knockdown, Plasmid Preparation, Over Expression, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

doi: 10.1073/pnas.1609552114

Figure Lengend Snippet: IGF2BP1 colocalizes with BCL11A in the ribosomes. RNA immunoprecipitation (RIP) using antibody against IGF2BP1 was performed to assess binding of RNAs to IGF2BP1 protein followed by RT-qPCR quantitation for (A) BCL11A transcripts. RIP was performed at culture day 14. Mean value ± SD of three independent donors for each condition is shown. (B) BCL11A stability analysis in IGF2BP1-OE versus empty vector control after actinomycin D treatment at culture day 14. Time course for RNA stability started by adding the transcription inhibitor actinomycin D (10 µg/mL) and cells were harvested at the indicated time points. Expression levels (percentage) were calculated considering the time point “0 h” as 100%. Mean value ± SD of two independent donors for each condition is shown. (C–H) Polysome profiling analysis of IGF2BP1-OE versus empty vector control at culture day 12. A representative polysome gradient (control versus IGF2BP1-OE) is shown in C. BCL11A mRNA was measured by RT-qPCR quantitation on the polysome fractions and mean value ± SD of two independent donors for each condition is shown in D. Western blot analysis of IGF2BP1 on representative polysome fractions from one representative donor is shown in E. Beta-globin (F), gamma-globin (G), and beta-globin + gamma-globin (H) mRNAs were measured by RT-qPCR quantitation on representative polysome profiling fractions. Results are shown from one representative donor. Molecular weight is shown in kilodaltons (kDa). P value was calculated using two-tailed Student’s t test. *P < 0.05. Experiments were performed in adult CD34(+) cells. BP1-RIP, IGF2BP1 RNA immunoprecipitation; IgG, immunoprecipitation with isotype control; Input, RNA sample before immunoprecipitation; OE, overexpression.

Article Snippet: RIP was performed on day 14 adult cells cultured from three independent donors transduced with IGF2BP1 lentiviral particles with antibody against IGF2BP1 (MBL International, RN007P) or rabbit IgG using RiboCluster Profiler RIP-Assay Kit (MBL International, RN1001) following manufacturer’s protocol.

Techniques: RNA Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Quantitation Assay, Plasmid Preparation, Control, Expressing, Western Blot, Molecular Weight, Two Tailed Test, Immunoprecipitation, Over Expression

Journal: Cell Research

Article Title: Comparison of viral RNA–host protein interactomes across pathogenic RNA viruses informs rapid antiviral drug discovery for SARS-CoV-2

doi: 10.1038/s41422-021-00581-y

Figure Lengend Snippet: a , b Loss-of-function screen to identify functional vRNA-interacting proteins. Candidate genes of vRNA-interacting proteins were knocked down using two distinct shRNAs in Caco-2 cells (Supplementary information, Fig. ) and subsequently infected with SARS-CoV-2 virus (MOI 0.05) ( a ); or knocked down in Huh7 cells (Supplementary information, Fig. ) and subsequently infected with ZIKV MR766 virus (MOI 0.5) ( b ). For a , viral loads in culture supernatants were quantified at 48 h post infection by qPCR (vRNA copy numbers). For b , intracellular ZIKV RNA levels in the gene-knockdown cells were quantified as a percentage relative to the control samples (ZIKV-infected Ctr cells with nontargeting scramble shRNAs) at 48 h post infection by qPCR, using GAPDH as internal control. Host candidates knocked down in both Caco-2 and Huh7 cells are indicated in red. Data are means ± SD, n = 3 independent biological samples. *** P < 0.001, ** P < 0.01, * P < 0.05. Two-tailed student’s t -test. c , d uvCLAP data for HSP90AB1 and IGF2BP1, aligned to the SARS-CoV-2 genome. HSP90AB1 ( c ) and IGF2BP1 ( d ) uvCLAP sequencing data are plotted across the SARS-CoV-2 genome. Zoomed-in views of the specific binding sites on the SARS-CoV-2 genomic RNA are shown below. The HSP90AB1-binding UA-rich sequences ( c ) and sequences matched to the known IGF2BP1-binding motif “CACA” ( d ) are highlighted and indicated in red rectangles. HSP90AB1, uvCLAP sequencing data for HSP90AB1; IGF2BP1, uvCLAP sequencing data for IGF2BP1; IgG, sequencing data of the IgG pull-down. e SARS-CoV-2 vRNA decay assay in Huh7 cells. Top, schematic diagram of the construction of the SARS-CoV-2 vRNA reporter (left) and the IGF2BP1 nonbinding control (right, Rluc). Middle, the schedules for RNA transfection and sample collection. The vRNA containing the 5′UTR, the coding sequence of the S protein, and the 3′UTR of SARS-CoV-2 (5′UTR-S-3′UTR) were transfected into WT cells, IGF2BP1-KO cells, and IGF2BP1 KO cells reexpressing IGF2BP1 (KO + IGF2BP1). The 5′UTR-S-3′UTR or Rluc RNA levels were quantified relative to GAPDH by qPCR at different hours post transfection; the percentage relative to 0 h post transfection cells is shown. f Detection of the S protein in WT, IGF2BP1-KO and KO + IGF2BP1 Huh7 cells. As in e , 5′UTR-S-3′UTR RNA of SARS-CoV-2 was transfected into cells. S protein levels in cells were detected using western blotting at 24 h post transfection. The upper panel shows a western blot for the S protein, GAPDH was used as sample loading control. Spike, western blot of S protein. The bar plot shows quantification of the band intensity from the western blot image. g In vitro translation assay of 5′UTR-S-3′UTR RNA. The 5′UTR-S-3′UTR RNA (500 ng) of SARS-CoV-2 was translated in rabbit reticulocyte lysates. Lysates without the 5′UTR-S-3′UTR RNA were used as a blank control for S protein detection in the in vitro translation system. Approximately 200 ng of purified IGF2BP1 was added into the lysates (+ IGF2BP1), or an equal volume of the buffer used with IGF2BP1 (–IGF2BP1). Non-specific, nonspecific protein band of the western blot. The bar plot shows quantification of the western blot. For e – g , n.s., not significant. *** P < 0.001, ** P < 0.01, * P < 0.05. Two-tailed student’s t -test. Data are means ± SD, n = 3 biologically independent samples.

Article Snippet: The KO cell lines were further confirmed at protein expression levels by western blotting using an antibody against IGF2BP1 (RN007P, MBL).

Techniques: Functional Assay, Infection, Virus, Knockdown, Control, Two Tailed Test, Sequencing, Binding Assay, Transfection, Western Blot, In Vitro, Purification

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: A GO annotation of 109 common tsRNA‐interacting proteins. B Effects of candidate 5′‐tsRNAs: tsGlnCTG, tsGlyGCC, tsMetCAT, and tsIleGAT on uncapped luciferase mRNA in an in vitro translation system ( n = 2–3, error bars represent SEM, and significance was calculated using unpaired t ‐test). B′ Effects of tsGlnCTG on capped GFP mRNA in an in vitro translation system ( n = 2–3, error bars represent SD, and significance was calculated using unpaired t ‐test). C Polysome profiles of different stem vs. differentiating states tested in this study. Gray areas indicate the fractions collected for sequencing. C′ tsRNA distribution among the polysome fractions from indicated cell states. D Workflow to identify proteomic and transcriptomic interactions of tsGlnCTG in pluripotency vs. differentiating mESCs. E False discovery rate (FDR) of peptides detected in LC‐MS/MS of biotinylated tsGlnCTG pulldown. F Western blot showing differential interaction of Igf2bp1, Ybx1, and Rpl10 with tsGlnCTG between LIF and RA conditions. G Overlap between transcripts that are associated with tsRNAs in RA condition and the transcripts that are affected upon ASO transfection in RA conditions. H Schematic of the experimental setup to identify protein‐mediated and complementarity‐based tsRNA target interaction. I qPCR analysis showing the enrichment of transcripts pulled down by tsGlnCTG in lysates devoid of proteins compared with normal lysate ( n = 2, error bars represent SEM, and significance was calculated using unpaired t ‐test). J Top 10 tsGlnCTG‐associated proteins in indicated cell states. Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001; n.s, non‐significant.

Article Snippet: This was followed by a transfer onto a nitrocellulose membrane and subsequently probed with antibodies against Igf2bp1 (8482, CST), Ybx1 (ab12148, Abcam), or Rpl10 (ab110371, Abcam).

Techniques: Luciferase, In Vitro, Sequencing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: A Merged triplicate Western immunoblots of Igf2bp1 and β‐actin in mESCs grown under LIF or RA conditions (top) and densitometric quantitation of the same (below) ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). B Validation of siRNA‐mediated knockdowns of Igf2bp1 in RA‐treated mESCs ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). B′ Expression of pluripotency genes in Igf2bp1 knockdown cells ( n = 3, error bars represent SD, and significance was calculated using Student's t ‐test). C Small RNAs identified (18–35 nt) from input and Igf2bp1 pulldown in RA‐treated mESCs. D qPCR validation of the tsGlnCTG enrichment upon Igf2bp1 pulldown in RA‐treated mESCs. E Binding curve showing the interaction of tsGlnCTG and Igf2bp1 (Kd = 33 nM). The intensities of the Igf2bp1 band were normalized to band intensity of the total Igf2bp1 protein used for pulldown. F Biological duplicate for c‐Myc quantitation from Igf2bp1 pulldown between LIF vs. RA conditions ( n = 2, for duplicate check Fig B). Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001; n.s, non‐significant.

Article Snippet: This was followed by a transfer onto a nitrocellulose membrane and subsequently probed with antibodies against Igf2bp1 (8482, CST), Ybx1 (ab12148, Abcam), or Rpl10 (ab110371, Abcam).

Techniques: Western Blot, Quantitation Assay, Biomarker Discovery, Expressing, Knockdown, Binding Assay

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. ( n = 2) error bars represent SEM. Quantification of the Igf2bp1‐bound c‐Myc mRNA between LIF‐ vs. RA‐treated mESCs ( n = 2; see Fig F for duplicate data). Association of c‐Myc transcript in different translating pools in RA‐treated mESCs as compared to LIF condition. ( n = 2), error bars represent SD, and significance was calculated by one‐tailed unpaired t ‐test. Relative enrichment of c‐Myc mRNA in translating (80S and polysome) and non‐translating (mRNPs) pools (fractionated from polysome profiling) between ASO‐treated and mock‐treated RA‐induced differentiating mESCs. ( n = 2), error bars represent SD, and significance was calculated using one‐tailed unpaired t ‐test. Relative levels of c‐Myc mRNA in ASO‐treated and mock‐treated RA‐induced differentiating mESCs compared with LIF‐treated mESCs. ( n = 3), error bars represent SD, and significance was calculated using one‐tailed unpaired t ‐test. Epistatic analysis of 5′‐tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity ( n = 3), error bars represent SD, and significance was calculated using unpaired t ‐test. Schematic representing tsRNA based c‐Myc transcript regulation. Data information: * P value < 0.05, ** P value < 0.01; *** P value < 0.001.

Article Snippet: This was followed by a transfer onto a nitrocellulose membrane and subsequently probed with antibodies against Igf2bp1 (8482, CST), Ybx1 (ab12148, Abcam), or Rpl10 (ab110371, Abcam).

Techniques: In Vitro, Binding Assay, One-tailed Test, Activity Assay

Journal: EMBO Reports

Article Title: Dynamic expression of tRNA‐derived small RNAs define cellular states

doi: 10.15252/embr.201947789

Figure Lengend Snippet: The dynamic expression of 5′‐tsRNAs plays a crucial role in modulating stem cell differentiation. During differentiation, 5′‐tsRNAs regulate the translation and/or the stability of several transcripts through its interaction with ribosomes, RNA‐binding proteins (ribonucleoproteins), such as IGF2BP1, or through direct sequence complementarity.

Article Snippet: This was followed by a transfer onto a nitrocellulose membrane and subsequently probed with antibodies against Igf2bp1 (8482, CST), Ybx1 (ab12148, Abcam), or Rpl10 (ab110371, Abcam).

Techniques: Expressing, Cell Differentiation, RNA Binding Assay, Sequencing

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: Cell division cycle‐associated 4 (CDCA4) interacts with IGF2BP1 and promotes the post‐transcriptional translation level of IGF2BP1. (a) The results of mass spectrometry analysis of differential fragments in A549 cells overexpressing CDCA4. (b) Reducing IGF2BP1 inhibits the clonogenic ability of A549 (mean ± SD, Student's t ‐test, * p < 0.05). (c) The downregulation of CDCA4 in A549 inhibits cell proliferation (mean ± SD, Student's t ‐test, *** p < 0.005). (d) The effect of decreasing IGF2BP1 expression on the phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway. (e) CO‐immunoprecipitation (IP) assay demonstrating the binding of CDCA4 and IGF2BP1 in A549 cells. (f) Western blotting analysis indicating changes in the IGF2BP1 protein expression in stable transgenic strains A549 and H1299 (mean ± standard deviation [SD], Student's t ‐test, * p < 0.05, ** p < 0.01). (g) The effect of upregulation versus inhibition of CDCA4 on the messenger RNA (mRNA) level of IGF2BP1. (h) Quantitative polymerase chain reaction (qPCR) analysis indicating the changes in the CDCA4 expression in A549 transfection with si‐IGF2BP1. (i) The effect of downregulation of IGF2BP1 in A549 on the expression of CDCA4 protein

Article Snippet: The supernatants were incubated overnight at 4°C with the protein G beads‐bound antibodies against igG (sc‐2025, Santa Cruz Biotechnology), antibodies against CDCA4 (11625‐AP, Proteintech), and antibodies against IGF2BP1 (55246‐1‐AP, Proteintech).

Techniques: Mass Spectrometry, Expressing, Immunoprecipitation, Binding Assay, Western Blot, Transgenic Assay, Standard Deviation, Inhibition, Real-time Polymerase Chain Reaction, Transfection

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: IGF2BP1 knockdown can alter the function of cell division cycle‐associated 4 (CDCA4). (a) Western blotting analysis showing the expression of IGF2BP1 protein in H1299, A549 stable transfer strain transfection with si‐IGF2BP1. (b) Effect of downregulation of IGF2BP1 on the proliferation efficiency of CDCA4 knockout cells H1299 and CDCA4 overexpressing cells A549 (mean ± standard deviation [SD], Student's t ‐test, *** p < 0.005). (c) Differences in the cell proliferation capacity as reflected by the real‐time cell analysis (RTCA) results (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01, *** p < 0.005). (d) The effect of the downregulation of IGF2BP1 on the phenotype of CDCA4 clone formation (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01). (e) EdU assay indicated the effect of IGF2BP1 knockdown on the proliferation ability of lung adenocarcinomas (LUAD) cells with high and low expression of CDCA4 (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01, *** p < 0.005)

Article Snippet: The supernatants were incubated overnight at 4°C with the protein G beads‐bound antibodies against igG (sc‐2025, Santa Cruz Biotechnology), antibodies against CDCA4 (11625‐AP, Proteintech), and antibodies against IGF2BP1 (55246‐1‐AP, Proteintech).

Techniques: Knockdown, Western Blot, Expressing, Transfection, Knock-Out, Standard Deviation, Cell Analysis, EdU Assay

Journal: Cellular and Molecular Immunology

Article Title: ZBP1 mediates interferon-induced necroptosis

doi: 10.1038/s41423-019-0237-x

Figure Lengend Snippet: ZBP1 is required for IFN-induced necroptosis. a The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-γ, IFN-γ + zVAD, or IFN-β, and cell survival was determined by measuring the ATP levels. b The p-RIPK3, RIPK3, and GAPDH protein levels in the indicated cells treated with or without IFN-γ for 36 h were measured by western blotting. The cells marked “ZBP1 sgRNA” were pooled ZBP1 KO cells, not isolated single clones of KO cells. c The ZBP1 and GAPDH protein levels in indicated cells treated with or without IFN-γ were measured by western blotting. d The ZBP1 and GAPDH protein levels in the indicated cells treated with or without IFN-γ, ActD, or CHX were measured by western blotting. e The ZBP1 and GAPDH protein levels in the indicated cells treated with or without IFN-γ, nifuroxazide, or a JAK inhibitor were measured by western blotting. For a, the data are from n = 3 independent experiments. The error bars indicate the mean ± s.e.m. of n = 3 independent experiments. A two-tailed Student’s t-test was applied to determine the P values. *P < 0.05, **P < 0.01. For b–e, the results shown are representative of the results of three independent experiments

Article Snippet: Antibodies against ZBP1 (M-300) (sc-67258), GAPDH (6C5), Myc (sc-40), HA (sc-805), and actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Knock-Out, Western Blot, Isolation, Clone Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: ZBP1 mediates interferon-induced necroptosis

doi: 10.1038/s41423-019-0237-x

Figure Lengend Snippet: RHIM-dependent interaction between ZBP1 and RIPK3 is essential for IFN-induced necroptosis. a Schematics illustrating the domains of ZBP1 and the ZBP1 RHIMmut mutant. b The indicated knockout L929 cell lines were infected with lentivirus expressing blank vector (Vector) or vectors encoding ZBP1 or ZBP1 RHIMmut, and cell survival at 24 h after infection was determined by measuring the ATP levels. c The p-RIPK3, RIPK3, p-MLKL, MLKL, and GAPDH protein levels in cells infected with lentivirus expressing blank vector or vectors encoding ZBP1 or its mutants were measured by western blotting. d A total of 293T cells were transfected with plasmids as indicated for 48 h. Total cell lysates (TCL) and anti-FLAG immunoprecipitates were immunoblotted to detect the indicated proteins. e A total of 293T cells were transfected with plasmids as indicated for 48 h. TCL and anti-FLAG immunoprecipitates were immunoblotted to detect the indicated proteins. f The indicated knockout L929 cell lines were transfected with plasmids expressing FLAG-RIPK3 and were then treated with or without IFN-γ or IFN-γ + zVAD for 36 h. TCL and anti-FLAG immunoprecipitates were immunoblotted to detect the indicated proteins. For a, the data are from n = 3 independent experiments. The error bars indicate the mean ± s.e.m. of n = 3 independent experiments. A two-tailed Student’s t-test was applied to determine the P values. **P < 0.01. For b, c, e, and f, the results shown are representative of the results of three independent experiments

Article Snippet: Antibodies against ZBP1 (M-300) (sc-67258), GAPDH (6C5), Myc (sc-40), HA (sc-805), and actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Knock-Out, Infection, Expressing, Plasmid Preparation, Western Blot, Transfection, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: ZBP1 mediates interferon-induced necroptosis

doi: 10.1038/s41423-019-0237-x

Figure Lengend Snippet: ZBP1 homointeraction is important in triggering RIPK3-dependent necroptosis. a, b WT L929 or RIPK3 KO L929 cells were infected with lentiviral vector to express proteins as indicated, and cell survival was determined at 36 h after infection by measuring the ATP levels. c A total of 293T cells were transfected with plasmids as indicated. TCL and anti-FLAG immunoprecipitates were immunoblotted to detect the indicated proteins. d A total of 293T cells were transfected with plasmids as indicated. TCL and anti-FLAG immunoprecipitates were treated with or without β-mercaptoethanol (β-Me) and immunoblotted to detect the indicated proteins. e WT L929 cells were infected with lentiviral vectors to express proteins as indicated and were then treated with PBS (ctrl), ethanol (ETOH), or 4-OHT for 12 h; cell survival was then determined by measuring the ATP levels. f WT L929 cells were infected with lentiviral vectors to express proteins as indicated. The FLAG and GAPDH protein levels were measured by western blotting. g A total of 293T cells were transfected with plasmids as indicated, and sequential immunoprecipitation with anti-Flag antibody and anti-HA antibody was performed. TCL and the anti-FLAG and sequential anti-HA immunoprecipitates were immunoblotted to detect the indicated proteins. For a, b, and e, the data are from n = 3 independent experiments. The error bars indicate the mean ± s.e.m. of n = 3 independent experiments. A two-tailed Student’s t-test was applied to determine the P values. **P < 0.01. For c, d, f, and g, the results shown are representative of the results of three independent experiments

Article Snippet: Antibodies against ZBP1 (M-300) (sc-67258), GAPDH (6C5), Myc (sc-40), HA (sc-805), and actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Infection, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: ZBP1 mediates interferon-induced necroptosis

doi: 10.1038/s41423-019-0237-x

Figure Lengend Snippet: The caspase-8-resistant RIPK3 mutant enhances IFN-induced necroptosis, and ZBP1 KO mice are protected against SIRS induced by TNF + IFN-γ. a Caspase-8 KO L929 cell lines were infected with lentivirus expressing blank vector or vectors encoding caspase-8 or caspase-8 C362S and were treated with the indicated reagents for 36 h. Cell survival was determined by measuring the ATP levels (bottom). Top: The caspase-8 and GAPDH protein levels in the cells described above were measured by western blotting. b FLAG-RIPK3, FLAG-RIPK3-D333E, and FLAG-ZBP1 were overexpressed in 293T cells and were then immunoprecipitated with anti-FLAG antibody. After incubation with recombinant active caspase-8 at 37 °C for 2 h, western blotting was used to detect the indicated proteins. c RIPK3 KO L929 cell lines were infected with lentivirus expressing blank vector or vectors encoding RIPK3 or RIPK3-D333E and were then treated with IFN-γ or IFN-β for 36 h. Cell survival was determined by measuring the ATP levels (bottom). Top: The RIPK3 and GAPDH protein levels in the cells described above were measured by western blotting. For a, c, the data are from n = 3 independent experiments. The error bars indicate the mean ± s.e.m. of n = 3 independent experiments. A two-tailed Student’s t-test was applied to determine the P values. **P < 0.01. For a–c, the results shown are representative of the results of three independent experiments. d WT mice were injected with 12 µg TNF, 30 µg IFN-γ, or TNF + IFN-γ via the tail vein. Mouse survival is presented as a Kaplan–Meier plot, and the log-rank (Mantel–Cox) test was performed. N = 6 mice for each group. The data are pooled from two independent experiments. *P < 0.05. e Age- and sex-matched WT and ZBP1 KO mice were injected with TNF + IFN-γ through the tail vein. Mouse survival is presented as a Kaplan–Meier plot, and the log-rank (Mantel–Cox) test was performed. N = 6 mice for each group. The data are pooled from two independent experiments. **P < 0.01

Article Snippet: Antibodies against ZBP1 (M-300) (sc-67258), GAPDH (6C5), Myc (sc-40), HA (sc-805), and actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Infection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Incubation, Recombinant, Two Tailed Test, Injection

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: Interaction of microRNA-222 (miR-222) with the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: miR-222 and U6 RNA levels in HCT-116 cells transfected with biotinylated miR-222 for 24 h. Values are means ± SE from 3 independent experiments (n = 4). *P < 0.01 compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B: levels of ZBP1, PLCγ1, claudin-1 (CCND1), and FZD7 mRNAs in the materials pulled down by biotin-miR-222 (left) and total input mRNAs (right) in cells described in A. Fzd7 served as a positive control.

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Transfection, Positive Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: Ectopically expressed microRNA-222 (miR-222) represses the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with pre-miR-222 as measured by quantitative PCR analysis. Values are means ± SE from independent experiments (n = 4). *P < 0.05, compared with cells transfected with control scrambled oligomer analyzed by one-way ANOVA followed by Duncan’s test. B: immunoblots of ZBP1, PLCγ1, and PCNA proteins in HCT-116 cells described in A. Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals as measured by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). D and E: changes in ZBP1, PLCγ1, and PCNA proteins in IEC-Cdx2L1 cells 48 h after transfection with pre-miR-222. Values are means ± SE (n = 3). Statistical test: means are compared with the scramble (cells exposed to pre-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) overexpression destabilizes the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with pre-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 3). *P < 0.01, compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Over Expression, Binding Assay, Transfection, Negative Control, Isolation, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) silencing enhances the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with anti-miR-222. Values are the means ± SE from 3 independent experiments (n = 4). *P < 0.05, compared with cells transfected with control oligomer (C-oligo). B: immunoblots of ZBP1, PLCγ, and PCNA proteins in cells described in A. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). Statistical test: means are compared with the scramble (cells exposed to Anti-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Transfection, Western Blot, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) silencing increases the stability of the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with anti-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 4). *P < 0.01, compared with cells transfected with C-oligo. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Transfection, Negative Control, Isolation, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222)-regulated expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) modulates rapid epithelial restitution after wounding. A: images of cell migration: a, 0 h after wounding in control cells (Con-0 h); b, 6 h after wounding in control cells (Con-6 h); c, 6 h after wounding in cells transfected with scramble oligo (C-oligo-6 h); d, 6 h after wounding in cells transfected with pre-miR-222 alone for 48 h (Pre-miR-222–6 h); e, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding ZBP1 (Pre-miR222 + ZBP1); f, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding PLCγ1 (Pre-miR222+ PLCγ1). Scale bar, 100 μm; magnification, ×100. B: summarized data showing rates of cell migration 6 h after wounding in cells described in A. Values are the means ± SE of data from 6 dishes and repeated 4 times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with pre-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. C: images of cell migration: a: 0 h after wounding in control cells; b: 6 h after wounding in control cells; c, 6 h after wounding in cells transfected with scramble oligo; d: 6 h after wounding in cells transfected with anti-miR-222 alone for 48 h; e: 6 h after wounding in cells cotransfected with anti-miR-222 and siZBP1 (Anti-miR222 + siZBP1); f: 6 h after wounding in cells cotransfected with anti-miR-222 and siPLCγ1 (Anti-miR222 + siPLCγ1); and g: 6 h after wounding in cells cotransfected with anti-miR-222, siZBP1 and siPLCγ1 (anti-miR222 + siZBP1 + siPLCγ1). Scale bar, 100 μm; magnification, ×100. D: summarized data showing rates of cell migration in cells described in C. Values are the means ± SE of data from 6 dishes and repeated four times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with Anti-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. E: immunoblot of PCNA protein in nonwounding and 0 h and 6 h after wounding (#1 and #2). Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels.

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Migration, Transfection, Plasmid Preparation, Western Blot

Journal: International journal of molecular sciences

Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.

doi: 10.3390/ijms242115600

Figure Lengend Snippet: Figure 2. IMP1 regulates the localization of SNHG15 at the cell protrusions. (A) FISH was per- formed to detect the subcellular localization of SNHG15 in WT BT-549 cells (upper panel) and SNHG15-expressing BT-549 cells (lower panel). The arrowheads indicate localized SNHG15 at the cell protrusions. Scale bar: 10 µm. (B) FISH indicated that, in comparison with WT MDA-MB-231 cells (upper panel), IMP1 expression greatly increased the localization of SHNG15 at cell protrusions (lower panel). Scale bar: 10 µm. (C) A bar graph indicates the percentage of protrusion-localized SNHG15 in tested cells. Localization was increased to 70% from 40% when ectopic IMP1 was ex- pressed. About 80–100 cells were counted in each group. ** p < 0.01. WT: cell origin. IMP1: cells expressing ectopic IMP1. Control: cells transfected with an empty plasmid. (D) RT-qPCR showed that IMP1 expression does not affect cellular levels of SNHG15.

Article Snippet: Primary antibodies against IMP1, GAPDH and nucleolin (NCL) were purchased from Cell Signaling Co. (Danvers, MA, USA); CDH2 and CDH1 antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Expressing, Comparison, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.

doi: 10.3390/ijms242115600

Figure Lengend Snippet: Figure 3. Localization of SNHG15 at the cell protrusions increases cell invasive potential. (A) The full-length SNHG15 and three dissected fragments of SNHG15 are separately tagged with six MS2 repeats. The relative position of the truncated SNHG15 is shown. The black box indicates the “ACACCC” motif for IMP1 binding. (B) FISH experiments showed that deletion of the “ACACCC” motif (Mut SNHG15) prevented SNHG15 from localizing at the cell protrusions. The arrowhead indicates cell-protrusion-localized SNHG15. Scale bar: 10 µm. (C) A bar graph shows that the cell population with protrusion-localized SNHG15 was largely decreased when the “ACACCC” motif was absent. ** p < 0.01. (D) Localization of the full-length and three truncated fragments of SNHG15 was determined by FISH. Results indicated that T1 and T3, but not T2, were still able to localize at the cell protrusions. About 80–100 cells were counted in each group. ** p < 0.01. (E) Transwell assays showed that, compared to the cells expressing full-length SNHG15, all three stable cell lines expressing truncated SNHG15 displayed lower invasive potentials. ** p < 0.01.

Article Snippet: Primary antibodies against IMP1, GAPDH and nucleolin (NCL) were purchased from Cell Signaling Co. (Danvers, MA, USA); CDH2 and CDH1 antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Binding Assay, Expressing, Stable Transfection