Journal: Journal of Cellular and Molecular Medicine

Article Title: Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

doi: 10.1111/jcmm.17946

Figure Lengend Snippet: The PCR primers used in this study.

Article Snippet: The protein samples were subjected to 12.5% SDS‐PAGE gel electrophoresis and transferred to the PVDF membrane in a BIO‐RAD transfer apparatus, then blocked in 5% skimmed milk for 1 h. The membrane was incubated overnight at 4°C with the primary antibody, including GAPDH (Proteintech, 10494‐1‐AP, 1:10,000), TH (Proteintech, 25859‐1‐AP, 1:5000), α‐syn (Cell Signalling Technology, D37A6, 1:1000), LC3 (Sigma, L7543, 1:1000), p62 (Sigma, P0067, 1:1000), ZFP27 (Antibodies, 2780268, 1:500), IGF2 (Immunoway, YN1761, 1:500), TfR1(abcam, ab214039, 1:1000), Gm4724 (biorbyt, A81254, 1:500).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

doi: 10.1111/jcmm.17946

Figure Lengend Snippet: ZFP27 and IGF2 may mediate iron accumulation exacerbating PD. (A) Heatmaps display the 37 differentially expressed genes (DEGs) in the PD model mouse and iron‐treated PD mice using RNA‐seq. (B) Venn diagram for DEGs including upregulated and downregulated genes by transcriptome analysis in mice. (C) QPCR determined four kinase‐related genes in the two groups. (D) Transcription factor Gm4724 in the SN showed no significant difference in the MPTP+iron group and MPTP group (western blot). The relative grey value of Gm4724 was calculated using Image J software. (E) Transcription factor ZFP27 in the SN was less in the MPTP+iron group compared to the MPTP group (western blot). The relative grey value of ZFP27 was calculated using Image J software. All data are presented as the mean ± SEM, ns p ≥ 0.05; * p < 0.05.

Article Snippet: The protein samples were subjected to 12.5% SDS‐PAGE gel electrophoresis and transferred to the PVDF membrane in a BIO‐RAD transfer apparatus, then blocked in 5% skimmed milk for 1 h. The membrane was incubated overnight at 4°C with the primary antibody, including GAPDH (Proteintech, 10494‐1‐AP, 1:10,000), TH (Proteintech, 25859‐1‐AP, 1:5000), α‐syn (Cell Signalling Technology, D37A6, 1:1000), LC3 (Sigma, L7543, 1:1000), p62 (Sigma, P0067, 1:1000), ZFP27 (Antibodies, 2780268, 1:500), IGF2 (Immunoway, YN1761, 1:500), TfR1(abcam, ab214039, 1:1000), Gm4724 (biorbyt, A81254, 1:500).

Techniques: RNA Sequencing Assay, Western Blot, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

doi: 10.1111/jcmm.17946

Figure Lengend Snippet: ZFP27 and IGF2 were obviously reduced with iron‐treated PD cell models. (A) The effects of various concentrations of MPP + , MPP + +iron (FAC), MPP + +DFO and MPP + +CQ on cell viability were determined using a CCK‐8 assay in SHSY5Y cells. (B–F) Western blot measuring TH, TfR1, LC3, p62, Beclin1, ZFP27 and IGF2 levels in SHSY5Y cells after MPP + , MPP + +CQ, MPP + +iron (FAC) or MPP + +DFO treatment. The relative quantification data of TH, TfR1, LC3, p62, Beclin1, ZFP27 and IGF2 relative to the internal control GAPDH were calculated using Image J software. All data are presented as the mean ± SEM. ns p ≥ 0.05; * p < 0.05.

Article Snippet: The protein samples were subjected to 12.5% SDS‐PAGE gel electrophoresis and transferred to the PVDF membrane in a BIO‐RAD transfer apparatus, then blocked in 5% skimmed milk for 1 h. The membrane was incubated overnight at 4°C with the primary antibody, including GAPDH (Proteintech, 10494‐1‐AP, 1:10,000), TH (Proteintech, 25859‐1‐AP, 1:5000), α‐syn (Cell Signalling Technology, D37A6, 1:1000), LC3 (Sigma, L7543, 1:1000), p62 (Sigma, P0067, 1:1000), ZFP27 (Antibodies, 2780268, 1:500), IGF2 (Immunoway, YN1761, 1:500), TfR1(abcam, ab214039, 1:1000), Gm4724 (biorbyt, A81254, 1:500).

Techniques: CCK-8 Assay, Western Blot, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

doi: 10.1111/jcmm.17946

Figure Lengend Snippet: IGF2 overexpression increased ZFP27 to activate autophagy, and IGF2 expression was inhibited by iron. (A) Negative control (NC) and IGF2 overexpression (IGF2 OE) by pLVX‐IRES‐mCherry showed the transfection efficiency in HEK293T cell lines. (B, C) After plasmid transfection in HEK293T cells, IGF2 OE significantly increased the mRNA levels of IGF2 and ZFP27 compared to NC. (D) IGF2 OE markedly increased the LC3II protein levels by western blot, and the band intensity of LC3II relative to GAPDH was analysed using Image J. (E) IGF2 mRNA levels in IGF2 overexpressed cells were restrained with iron (FAC). All data are presented as the mean ± SEM, ns p ≥ 0.05; * p < 0.05.

Article Snippet: The protein samples were subjected to 12.5% SDS‐PAGE gel electrophoresis and transferred to the PVDF membrane in a BIO‐RAD transfer apparatus, then blocked in 5% skimmed milk for 1 h. The membrane was incubated overnight at 4°C with the primary antibody, including GAPDH (Proteintech, 10494‐1‐AP, 1:10,000), TH (Proteintech, 25859‐1‐AP, 1:5000), α‐syn (Cell Signalling Technology, D37A6, 1:1000), LC3 (Sigma, L7543, 1:1000), p62 (Sigma, P0067, 1:1000), ZFP27 (Antibodies, 2780268, 1:500), IGF2 (Immunoway, YN1761, 1:500), TfR1(abcam, ab214039, 1:1000), Gm4724 (biorbyt, A81254, 1:500).

Techniques: Over Expression, Expressing, Negative Control, Transfection, Plasmid Preparation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

doi: 10.1111/jcmm.17946

Figure Lengend Snippet: A proposed model for iron inhibition of autophagy in PD mice. In PD mice, accumulated iron suppressed kinase activator IGF2 and transcription factor ZFP27 to reduce LC3‐induced autophagy, finally increased the death of dopamine neurons and α‐syn accumulation in substantia nigra.

Article Snippet: The protein samples were subjected to 12.5% SDS‐PAGE gel electrophoresis and transferred to the PVDF membrane in a BIO‐RAD transfer apparatus, then blocked in 5% skimmed milk for 1 h. The membrane was incubated overnight at 4°C with the primary antibody, including GAPDH (Proteintech, 10494‐1‐AP, 1:10,000), TH (Proteintech, 25859‐1‐AP, 1:5000), α‐syn (Cell Signalling Technology, D37A6, 1:1000), LC3 (Sigma, L7543, 1:1000), p62 (Sigma, P0067, 1:1000), ZFP27 (Antibodies, 2780268, 1:500), IGF2 (Immunoway, YN1761, 1:500), TfR1(abcam, ab214039, 1:1000), Gm4724 (biorbyt, A81254, 1:500).

Techniques: Inhibition

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

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 1. IGF2BP2 expression in LPS activated human macrophages. A) IGF2BP2 mRNA level in HMDMs treated by saline (Control) or LPS (100 ng mL−1) at indicated time. B) IGF2BP2 protein expression in HMDMs treated by normal saline or LPS (100 ng mL−1) for 4 and 8 h. C) Statistically analysis of (B). D) IGF2BP2 protein expression in HMDMs treated by saline or LPS (100 ng mL−1) for 24, 36, and 48 h. E) Statistically analysis of (D). F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP). The mRNA levels in the Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control group. p-values were determined by using two-way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Quantitative RT-PCR, Western Blot

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

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 2. IGF2BP2 expression in LPS activated mouse macrophages. A) IGF2BP2 mRNA level in BMDMs treated by saline (Control) or LPS (100 ng mL−1) at the indicated time. B) IGF2BP2 protein expression in BMDMs treated by normal saline or LPS (100 ng mL−1) at the indicated time points, anti-IGF2BP2 antibody was purchased from Proteintech (11601- 1-AP); qPCR and western blot biological replicates from three different donors; IGF2BP2 mRNA levels in Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, versus the Control group. p-values were determined by using a two- way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Western Blot

Journal: PLoS Genetics

Article Title: Distinct Requirements for Cranial Ectoderm and Mesenchyme-Derived Wnts in Specification and Differentiation of Osteoblast and Dermal Progenitors

doi: 10.1371/journal.pgen.1004152

Figure Lengend Snippet: (A, B) RT-PCR for individual Wnt ligands was performed on cDNA from purified mouse embryonic cranial mesenchyme and surface ectoderm. (C, D G, H) Indirect immunofluorescence with DAPI counterstained nuclei (blue), (E) in situ hybridization, or immunohistochemistry (F, I) was performed on coronal mouse embryonic head sections. (G, H, I) Boxes indicate region in insets at higher magnification. White arrowheads indicate co-expression of (G) Wls/Runx2 or (D,H) Lef1/Runx2, (I) red arrowheads indicate osteoblast progenitors, and blue arrowheads indicate dermal progenitors. (F–I) White hatched lines demarcate ectoderm from mesenchyme. (J) Summary scheme of E12.5 supraorbital cranial mesenchyme. (J) Embryonic axes, figure depicts lateral view of embryonic head, region of interest in sections used in figures are shown. Scale bars represent 100 µm.

Article Snippet: Primary antibodies for Runx2, Sox9, Twist2, Lef1, Osx, Msx2, Ki67, IGF2, Wls, and β-catenin (goat anti-Runx2; 1∶20, R&D Biosystems; rabbit anti-Sox9; 1∶100; Millipore; mouse anti-Twist2, 1∶500, Santa Cruz; rabbit anti-Lef1, 1∶100, Abcam; rabbit anti-Osx, 1∶400, Abcam; mouse Msx1/2, 1∶50, DSHB; activated Caspase3, 1∶250, Abcam; rabbit Ki67; 1∶500 Abcam; rabbit IGF2 1∶400, Cell Signaling); rabbit anti-Wls, 1∶2000, gift from Richard Lang; mouse β-catenin 1∶100 BD Biosciences) were used for indirect immunofluorescence and immunohistochemistry.

Techniques: Reverse Transcription Polymerase Chain Reaction, Purification, Immunofluorescence, In Situ Hybridization, Immunohistochemistry, Expressing

Journal:

Article Title: IGF-2 and FLT-1/VEGF-R1 mRNA levels reveal distinctions and similarities between congenital and common infantile hemangioma

doi: 10.1203/PDR.0b013e318163a243

Figure Lengend Snippet: IGF-2 protein localized in relation to blood vessels in common and congenital hemangioma. A–C) CD31 immunostaining of frozen sections highlights blood vessels in proliferating common hemangioma (A), NICH (B) and RICH (C). Nuclei are stained blue with DAPI. D–E) Common hemangioma sections double-labeled with anti-IGF2 (green) and anti-CD31 (red). Nuclei stained with DAPI (blue). Panel D, a proliferating phase hemangioma while Panel E, involuting phase hemangioma. F) RICH double-labeled with anti-IGF2 (green) and anti-CD31 (red). G) NICH stained with anti-IGF2 (green). H) Pyogenic granuloma stained with anti-IGF2 (green) as negative control. Scale bars are 50 µm. Panel A is 200X magnification; Panels B, C, G and H, 100X magnification; Panels D–F, 300X magnification.

Article Snippet: Indirect immunofluorescence Cryosections ( 4 μm ) of the tumors (proliferating and involuting infantile hemangioma), RICH, NICH and pyogenic granulomas were fixed with cold acetone for 15 min. After washing for 5 min in PBS, and incubating with species appropriate blocking serum, the following primary antibodies were used: mouse anti-human IGF2 mAb 1:50 (R&D Systems, USA), mouse anti-human CD31 mAb 1:50 (DAKO, Carpinteria, CA, USA), goat anti-human CD31 antibody 1:50 (Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Immunostaining, Staining, Labeling, Negative Control