Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: Hypoxia led to abnormal mitochondrial morphology and function. (A) Immunofluorescence staining of mitochondrial morphology in HN6 cells. The cells were cultured under normoxia, hypoxia (48 h), or CCCP (2 h), and the mitochondria were labelled with HSP60. (B) Quantitative assessment of mitochondrial morphology in (A) using the Image J software. Five images per group were analysed randomly. (C) TEM images of the mitochondrial ultra‐structure in SCC25 and CAL27 cells under normoxia versus hypoxia. (D) JC‐1 aggregate/monomer ratios were measured by flow cytometry under normoxia, hypoxia, or CCCP treatment in CAL27 cells. (E) Quantification of mitochondrial depolarization. Median fluorescence intensity ratios of JC‐1 monomers versus aggregates were calculated and normalized against the mean values of the normoxia group. (F) MitoSOX assay to assess the mitochondrial superoxide levels in CAL27 cells. (G) Flow cytometric analysis of superoxide production after normoxia or hypoxia (48 h) treatment. (H) Seahorse extracellular flux assay under normoxia versus hypoxia in SCC25 cells. (I) Quantitative metrics of basal respiration, ATP production, maximal respiration, and spare capacity. (J) ATP production in CAL27 cells under hypoxia versus normoxia (48 h). Statistical analysis: Unpaired two‐tailed Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data are expressed as the mean ± standard deviations (SD).

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Immunofluorescence, Staining, Cell Culture, Software, Flow Cytometry, Fluorescence, Mitosox Assay, XF Assay, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: Hypoxia caused mitophagy, which does not involve fusion with lysosomes. (A) Spatial clustering of heterogeneous mitophagy and hypoxia profiles was observed across tumour and adjacent tissues in 10× Visium spatial transcriptomic slides. (B) Western blot analysis of the expression levels of PINK1, P62, LC3B, and p‐Ub. Proteins were extracted from SCC25 cells subjected to normoxia, hypoxia, or lactic acid for 48 h; starvation for 12 h; or CCCP treatment for 2 h. (C) Immunofluorescence staining of HN6 cells using MitoTracker and LC3B. (D) Quantitative analysis of LC3B‐MitoTracker co‐localization in (C), with n = 10 cells per group. (E) TEM images of the mitochondrial autophagosomes in SCC25 and CAL27 cells. (F) Quantification of mitochondrial autophagosomes per cell in (E), analysed in n = 10 cells per group. (G) HN6 cells subjected to normoxia, hypoxia (48 h), or CCCP (2 h) were stained with MitoTracker and LAMP1 to assess the degree of mitochondrial–lysosomal fusion. Statistical analysis: Unpaired two‐tailed Student's t test. **** p < 0.0001. The data are expressed as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: Hypoxia damaged lysosomal homeostasis. (A) Schematic illustration of mitochondrial autophagosome‒lysosome fusion and degradation. (B) Western blot analysis of the expression levels of LAMP1 and CTSD. Proteins were extracted from SCC25 and CAL27 cells subjected to normoxia and hypoxia for 48 h. (C) Representative TEM images of lysosomes from SCC25 and CAL27 cells subjected to hypoxia and normoxia. (D) Confocal microscopy analysis of LAMP1 in HN6 cells. (E) Lysosomal diameter distribution from three independent experiments ( n > 80 in each group). (F) The frequency distribution of lysosomal diameters indicated that the number of lysosomes (diameter < 800 nm) in hypoxic cells decreased, whereas the number of large lysosomes (diameter > 800 nm) increased significantly. (G) LysoTracker Red assay of HN6 cells exposed to normoxia or hypoxia for 48 h. (H) Quantification of the number of LysoTracker‐positive points in Figure G. Ten fields were randomly selected from each group for analysis. (I) Lysosomal pH was determined using Lysosensor yellow/blue DND‐160. Each point represents the lysosomal pH from an independent experiment. (J) Co‐localization of LAMP1 and TOM70 in HN6 cells. Statistical analysis was performed using an unpaired two‐tailed Student's t test. ** p < 0.01, **** p < 0.0001. The data are expressed as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Western Blot, Expressing, Confocal Microscopy, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: Hypoxia promoted the release of mitochondrial components through EV. (A) Co‐localization of RAB27A and TOM70 in HN6 cells under normoxia or hypoxia for 48 h. Co‐localization levels were quantified using the ImageJ software. (B) Fluorescence co‐localization of CD81 and HSP60 in HN6 cells after 48 h of normoxia/hypoxia. (C) HSP60 and CD81 co‐localization levels in hypoxic regions of HNSCC samples determined by multiplex immunohistochemistry (mIHC) staining. The images represent distinct regions in the same patient. (D) Mitochondrial proteins were significantly enriched in EV, as analysed by gene ontology (GO) enrichment. (E) Heatmap depicting mitochondria‐related differentially expressed proteins in EV under normoxic and hypoxic conditions. (F) Proportion of mitochondrial and non‐mitochondrial proteins in the EV. (G) Western blot analysis of EV purified from SCC25 cell culture supernatant. The mitochondrial proteins COXIV, ACO2, HSP60, and CS were detected, with ALIX serving as an EV loading control. (H) qPCR quantification of mtDNA in EV. Statistical analysis: unpaired two‐tailed Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data are presented as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Software, Fluorescence, Multiplex Assay, Immunohistochemistry, Staining, Western Blot, Purification, Cell Culture, Control, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: ATP6V1A overexpression led to a decrease in mitochondrial components in EV. (A) HN6 cells overexpressing ATP6V1A were exposed to hypoxia for 48 h and co‐stained with LAMP1 and HSP60. (B) Fluorescence co‐localization of CD81 and HSP60 in ATP6V1A‐overexpressing HN6 cells after 48 h of hypoxia. (C) EV from SCC25 cell supernatants (hypoxia‐treated and ATP6V1A‐overexpressing cells) were analysed by western blot. Mitochondrial proteins (COXIV, ACO2, HSP60, and CS) were detected, with ALIX serving as a loading control. (D) qPCR quantification of mtDNA in EV. (E) Western blot analysis of EV purified from HN6, SCC25, and CAL27 cells under normoxic or hypoxic conditions. Syntenin was used as a loading control. (F) HN6 cells with LC3B knockdown were stained with MitoTracker to visualize mitochondrial morphology. (G) EV purified from SCC25 cell supernatants (hypoxia‐treated, LC3B‐knockdown) were analysed by western blot. Mitochondrial proteins (TOM70, COXIV, and CS) were detected using syntenin as the loading control. (H) qPCR quantification of mtDNA in EV. Statistical analysis: Unpaired two‐tailed Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The data are presented as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Over Expression, Staining, Fluorescence, Western Blot, Control, Purification, Knockdown, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: The vesicle transporter proteins VAMP4 and STX8 mediated ASM. (A) Schematic representation of MVB isolation via density gradient sucrose centrifugation. (B) KEGG pathway enrichment analysis of differentially expressed proteins among MVB members. (C) The predicted interaction model of VAMP4 and STX8 visualized using PyMOL. The red regions denote the interface‐binding domains. (D) Co‐immunoprecipitation (co‐IP) assays confirmed the VAMP4‐STX8 interaction in HN6 and CAL27 cells. (E) Co‐IP analysis confirmed VAMP4‐STX8 complex formation within MVB isolated from HN6 and CAL27 cells. (F) Immunofluorescence staining for VAMP4, STX8, and CD63 in HN6 cells. (G) Immunofluorescence staining for VAMP4, STX8, CD81, and LC3B in HN6 cells. (H) Western blot analysis of VAMP4 and STX8 expression in HN6 and CAL27 cells under normoxia and hypoxia (0–48 h). (I) Hypoxia‐treated HN6 cells transfected with siVAMP4/siSTX8 were subjected to mIHC for HSP60, LC3B, and CD81 co‐localization. (J) Western blot quantification of mitochondrial proteins (COXIV and HSP60) in EV purified from SCC25 cells under hypoxia/normoxia with siVAMP4/siSTX8 knockdown. ALIX was used as the loading control. (K) qPCR quantification of mtDNA in EV isolated from SCC25 cells. (L) Western blot analysis of apoptosis‐related proteins (Bax and Caspase‐9) in SCC25 cells subjected to hypoxia/normoxia with mitophagy inhibition (3MA) or siVAMP4/siSTX8 transfection. (M) Western blot analysis of mitochondrial proteins (COXIV, ACO2, HSP60, and CS) in EV from HN6 cells transfected with VAMP4/STX8 truncation plasmids under hypoxia. ALIX was used as the loading control. (N, O) RT‐qPCR quantification of mtDNA in EV from HN6 cells transfected with VAMP4/STX8 truncation plasmids. Statistical analysis: unpaired two‐tailed Student's t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data are presented as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Isolation, Centrifugation, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Transfection, Purification, Knockdown, Control, Inhibition, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: VAMP4/STX8 Mediate the Autophagic Secretion of Mitochondria and Promote TAMs Polarization in HNSCC

doi: 10.1002/jev2.70276

Figure Lengend Snippet: STX4 and SNAP23 mediate EV release. (A) mIHC analysis of VAMP4, STX4, SNAP23, and CD81 co‐localization in hypoxia‐treated HN6 cells. TEM images of EV (B) and MVB (C) isolated from HN6 cells transfected with siSTX4 or siSNAP23. (D) NTA of EV size and quantity in SCC25 cells transfected with siSTX4/siSNAP23. EV were isolated from 2 × 10 6 cells/group. (E) BCA assay quantification of protein concentrations in EV collected from equal numbers of cells. (F) Western blot analysis of EV markers (ALIX, TSG101, and RAB5) in CAL27 cells transfected with siSTX4/siSNAP23. (G) Hypoxia‐treated SCC25 cells were transfected with siSTX4/siSNAP23. Western blot analysis of mitochondrial proteins (ACO2 and HSP60) in EV. ALIX was used as the loading control. (H) qPCR quantification of mtDNA in EV derived from CAL27 cells transfected with siSTX4/siSNAP23. (I) A schematic diagram of proposed mechanism. Statistical analysis: unpaired two‐tailed Student's t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data are presented as the mean ± SD.

Article Snippet: The SCC25 and CAL27 cell lines were obtained from ATCC (USA), and the UMD School of Dentistry (USA) provided the HN6 cell line.

Techniques: Isolation, Transfection, BIA-KA, Western Blot, Control, Derivative Assay, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Monocyte Chemoattractant Protein 1 Promotes VEGF-A Expression in OSCC by Activating ILK and MEK1/2 Signaling and Downregulating miR-29c

doi: 10.3389/fonc.2020.592415

Figure Lengend Snippet: Clinical significance of MCP-1 in angiogenesis of oral squamous cell carcinoma. (A–C) OSCC tissue and adjacent normal tissue data from the Oncomine database ( https://www.oncomine.org ) were analyzed for MCP-1, VEGF-A, and ANGPT2 expression. (D) IHC photographs of tissue array sections (OR601) immunostained with anti-MCP-1 or anti-VEGF-A antibody. (E, F) IHC results were scored on a scale of 1–5 for staining intensity. Levels of MCP-1 and VEGF-A expression correlated with OSCC clinical grade. **p < 0.005 , ***p < 0.0005 compared with controls. (A–C, E) are box-and-whisker plots that plot outliers as individual points (dots).

Article Snippet: The human OSCC cell line used in this study was SCC4, and was purchased from the Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan).

Techniques: Expressing, Staining, Whisker Assay

Journal: Frontiers in Oncology

Article Title: Monocyte Chemoattractant Protein 1 Promotes VEGF-A Expression in OSCC by Activating ILK and MEK1/2 Signaling and Downregulating miR-29c

doi: 10.3389/fonc.2020.592415

Figure Lengend Snippet: MCP-1 promotes human OSCC cell angiogenesis through the VEGF-A-dependent pathway. Cells were incubated with MCP-1 (0–50 ng/ml) for 24 h and VEGF-A expression was determined by Western blot (A) , RT-qPCR (B) , and ELISA assays (C) . The CM was collected and used to treat the EPCs for 24 h. We used the tube formation assay to examine capillary-like structure formation (D) and the Transwell assay to examine cell migration (E) . * p < 0.05 compared with controls; # p < 0.05 compared with MCP-1–treated controls. Each experimental procedure was independently repeated three times, with similar results.

Article Snippet: The human OSCC cell line used in this study was SCC4, and was purchased from the Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan).

Techniques: Incubation, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Transwell Assay, Migration