Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: pmRFP C1-CHMP4B , Sino Biological , HG14022-ANR.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: Uncropped images of immunoblots displayed in main figures. (A) Uncropped version of immunoblot displayed in main B . Membranes were incubated with anti-V5 (CST) and GAPDH (Abcam) antibodies prior to development. Inset shows cropped area shown in main figure. (B-E) Uncropped versions of immunoblots displayed in main A . The membranes were cut horizontally prior to incubation with antibodies for ALIX (CST), V5 (CST), GAPDH (Abcam), GM130 (BD Biosciences) and CHMP4B (Atlas Antibodies/Sigma).

Article Snippet: IF detection was performed using anti-CHMP4B (Atlas Antibodies/Sigma, Cat.: HPA051751, Lot No.: R67106, dilution 1:300, anti-V5 (CST, Cat.: 13202S, Lot No.: 2, dilution 1:150) and secondary antibodies; Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, (Thermo Fisher Scientific, Cat.: A32728, Lot No.: RJ243424, dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, Cat.: A32731, Lot No.: RJ243417, dilution 1:1000).

Techniques: Western Blot, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in . IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in Supplementary Figure S3. IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm

Article Snippet: IF detection was performed using anti-CHMP4B (Atlas Antibodies/Sigma, Cat.: HPA051751, Lot No.: R67106, dilution 1:300, anti-V5 (CST, Cat.: 13202S, Lot No.: 2, dilution 1:150) and secondary antibodies; Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, (Thermo Fisher Scientific, Cat.: A32728, Lot No.: RJ243424, dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, Cat.: A32731, Lot No.: RJ243417, dilution 1:1000).

Techniques: Western Blot, Isolation, Expressing, Negative Control, Marker, In Vivo, Staining

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: A novel biochemical interaction between MYH10 and ESCRT-III. (A) Co-immunoprecipitation analysis of MYH10 and ESCRT-III subunits (CHMP2B WT , CHMP2B intron5 , CHMP4A, CHMP4B, and CHMP4C). HEK293 cells were transfected with vectors expressing proteins tagged with Flag-MYH10 and green fluorescent proteins (GFP, GFP-CHMP2B WT , -CHMP2B intron5 , -CHMP4A, -CHMP4B, or -CHMP4C) Co-immunoprecipitation with anti-Flag agarose beads and western blotting were done with anti-Flag antibody or anti-GFP antibody. (B) Cellular localization of MYH10 with ESCRT-III subunits. U2OS cells stably expressing GFP-MYH10 were transiently transfected with mRFP-CHMP2B WT , -CHMP2B intron5 , -CHMP4A, -CHMP4B, or -CHMP4C. Scale bar: 20 μm. (C) Interaction of endogenous proteins CHMP4B and MYH10. HEK293T cell lysates were immunoprecipitated with anti-CHMP4B antibody; nonimmune IgG served as negative control. Western blotting was done with antibodies against MYH10, MYO5B, or CHMP4B. (D) Schematic diagram of the domain structure of CHMP4B. CHMP4B contains six α-helices and a microtubule-interacting and transport (MIT)-interacting motif (MIM) that mediates VPS4 binding. (E) Immunoprecipitation analysis of the interaction between endogenous MYH10 and full-length (FL) or various deletion mutants of CHMP4B. Flag-tagged proteins (Flag-CHMP4B full length, N-terminal half of CHMP4B (residues 1–118), or C-terminal half of CHMP4B (residues 119–224) were transfected in HEK293T cells, and immunoprecipitation was done with anti-Flag agarose beads. Western blotting was done with anti-Flag antibody or anti-MYH10 antibody. (F) Schematic diagram of the domain structure of MYH10. MYH10 has a head-like domain and coiled-coil domain. (G-H) Immunoprecipitation analysis of the interaction between CHMP4B and various deletion mutants of MYH10. Flag-CHMP4B and MYC/cMyc-tagged proteins (M1-, M2-, M3-, M4-, N-terminal half of M4-, or C-terminal half of M4-MYC) were transfected into HEK293T cells and co-immunoprecipitated with anti-Flag agarose beads. Western blotting was done with anti-Flag antibody or anti-MYC antibody.

Article Snippet: pEGFP C1-CHMP4B , Sino Biological , HG14022-ANG.

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Stable Transfection, Negative Control, Binding Assay

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 is involved in phagophore closure. (A) Representative immunofluorescence images showing completed autophagosomes (completely sealed autophagosomes) in WT or MYH10 KO U2OS cells stably expressing mRFP-STX17. The cells were stained with antibodies against LC3 and CANX (calnexin), an endoplasmic reticulum (ER) marker, and were subjected to amino acid starvation in the absence or presence of CHMP4B knockdown. White arrows indicate LC3- and STX17-positive completely closed autophagosomes. Scale bar: 20 μm. (B-C) Number and relative percentage of STX17-positive completed autophagosomes, which was calculated as the number of STX17-positive autophagosomes divided by the total number of LC3-positive autophagosomes. The cells were counted with the ITCN plugin using FIJI. Values are mean ± SEM of >16 randomly selected cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. (D) U2OS cells stably expressing YFP-PRKN and mRFP-STX17 were stained with MitoTracker (blue) after treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 20 μM) for 2 h in the absence or presence of CHMP4B knockdown. White arrows indicate PRKN- and STX17-positive completely closed mitophagosomes. Scale bar: 20 μm. (E-F) Number and relative percentage of PRKN- and STX17-positive mitophagosomes. which was calculated as the number of STX17-positive mitophagosomes divided by the total number of PRKN-positive damaged mitochondria. WT, wild-type; KO, knockout. Values are mean ± SEM of n > 17 randomly selected cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.

Article Snippet: pEGFP C1-CHMP4B , Sino Biological , HG14022-ANG.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Marker, Knock-Out

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 recruits ESCRT-III to PRKN-positive damaged mitochondria to mediate mitophagosome sealing. (A) U2OS cells stably expressing YFP-PRKN and mCherry-CHMP4B were stained with MitoTracker (blue) and treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 20 μM) and bafilomycin A 1 (BafA1, 100 nM) for 2 h. White arrows indicate PRKN- and CHMP4B-positive mitochondria. Scale bar: 20 μm. (B-C) Number and relative percentage of PRKN- and CHMP4B-positive mitophagosomes, which was calculated as the number of CHMP4B-positive mitophagosomes divided by the total number of PRKN-positive damaged mitochondria. Values are mean ± SEM of n > 16 randomly selected cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by paired two-tailed t test. (D) Representative transmission electron microscopy (TEM) images of mitochondria-containing multilamellar structures in WT or MYH10 KO U2OS cells. The cells were treated with CCCP (20 μM) for 2 h in the absence or presence of CHMP4B knockdown. Yellow arrows indicate multilamellar structures; M indicates mitochondria. Scale bar: 2 μm.

Article Snippet: pEGFP C1-CHMP4B , Sino Biological , HG14022-ANG.

Techniques: Stable Transfection, Expressing, Staining, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: pEGFP C1-CHMP4B , Sino Biological , HG14022-ANG.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Primer sequences.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Risk model and survival analysis for prognostic evaluation of OS Patients. (A,B) LASSO selection analysis plots of OS-related genes within the greenyellow module. (C) The ROC curve. (D) Kaplan-Meier survival analysis results, showing gene survival analysis curves significantly associated with the prognosis of OS patients (including CHMP4C, FAM222B, PROSER2, SNORA12, ZNF200).

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Selection

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Expression of CHMP4C and β-catenin in hFOB1.19, U2OS, HOS, and MG63 cells. (A) RT-qPCR analysis of CHMP4C and β-catenin mRNA expression. (B–D) : Immunofluorescence staining to detect the expression of CHMP4C, p-GSK3β, and β-catenin proteins. The Merge image is on the left side, DAPI (blue) is above on the right side, and FITC (green) is below on the right side. * p < 0.05, *** p < 0.001, **** p < 0.0001 compared with hFOB1..19 group. ## p < 0.01, ### p < 0.001 compared with U2OS group. ^ p < 0.05, ^^^ p < 0.001 compared with HOS group.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Impacts of overexpression and knockdown of CHMP4C on the expression of GSK3β, p-GSK3β, and β-catenin in MG63 cells. (A) RT-qPCR analysis of CHMP4C, GSK3β, and β-catenin mRNA expression in MG63 cells. (B–E) : Immunofluorescence staining to assess the expression of CHMP4C, GSK3β, p-GSK3β, and β-catenin proteins in MG63 cells. The Merge image is on the left side, DAPI (blue) is above on the right side, and FITC (green) is below on the right side. ** p < 0.01, *** p < 0.001 compared with OE-NC / IN-NC group.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Over Expression, Knockdown, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Assessment of the impacts of CHMP4C overexpression and knockdown on the proliferation and migration of OS Cells. (A) CCK-8 assay for MG63 cell proliferation. (B) Colony formation assay for quantifying the number and efficiency of MG63 cell colonies. (C) Transwell assay for assessing the migration of MG63 cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with OE-NC / IN-NC group.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Over Expression, Knockdown, Migration, CCK-8 Assay, Colony Assay, Transwell Assay

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Impacts of CHMP4C overexpression and knockdown on the growth of nude mouse OS and the expression of downstream pathway genes. (A,B) : Effects of CHMP4C overexpression and knockdown on the weight and volume of nude mouse OS. (C) RT-qPCR analysis of mRNA expression levels of CHMP4C, GSK3β, and β-catenin in tumor tissues. (D) Immunofluorescence staining analysis of protein expression levels of CHMP4C, GSK3β, p-GSK3β, and β-catenin in tumor tissues. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with OE-NC / IN-NC group.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Over Expression, Knockdown, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation

doi: 10.3389/fphar.2024.1399625

Figure Lengend Snippet: Diagram of the Molecular Mechanisms underlying CHMP4C CHMP4C within OS cells. CHMP4C inhibits the expression of GSK3β in OS cells, while upregulating the expression of pGSK3β and β-catenin. The phosphorylation of GSK3β further promotes β-catenin signaling transduction, activating the pGSK3β/β-catenin signaling pathway and promoting the proliferation and migration of OS cells.

Article Snippet: Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C.

Techniques: Expressing, Transduction, Migration