Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: Differential roles of P2X4 and P2X7 receptors in inducing sEV production under hypoxic conditions. CTL, CR4 (knock-down for the expression of P2X4) and CR7 (knock-down for the expression of P2X7) cells were grown under hypoxic conditions (1% O2) for a duration of 48 h prior to conducting analyses. a Size distribution and concentration of EVs purified from CTL, CR4 or CR7 cells grown under hypoxia, assessed by NTA (n = 12 independent experiments for CTL and CR4, n = 6 independent experiments for CR7). b Relative concentrations of sEVs per mL of supernatants, expressed relatively to that for CTL, in the same conditions than in a. The concentration of sEV produced by CR4 cells was significantly higher as compared to CTL (**, p <0.01). NS stands for not statistically different. c Protein concentrations of sEV extracts from same conditions than in a. There was a significantly higher protein concentration of sEVs extracts from CR4 cells, as compared to CTL (*, p <0.05) or to CR7 (*, p <0.05) cells. NS stands for not statistically different. d Relative concentration of sEVs per mL of supernatants coming from CTL cells treated with either 5-BDBD (P2X4 antagonist, 5µM) or A438079 (P2X7 antagonist, 10 µM) and expressed relatively to the CTL condition (vehicle). There was a significantly higher concentration of sEV produced by cells submitted to the P2X4 antagonist as compared to CTL (*, p <0.05) but not with cells treated with the P2X7 antagonist. NS stands for not statistically different. e Mean size (in nm) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in same conditions than in a. EVs produced by CR4 cells were significantly smaller than CTL (*, p <0.05). NS stands for not statistically different. f Zeta potential (in mV) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in similar conditions than in a. There was no difference between the three groups (n = 4 independent experiments). g Proportion of CD9+, CD63+ and CD81+ sEV purified from CR4 cells supernatants, assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 12 independent experiments). There was a significantly higher proportion of CD9+ sEV produced by CR4 cells compared to CTL cells (*, p <0.05). h Proportion of CD9+, CD63+ and CD81+ sEVs purified from supernatants of CTL cells treated with 5-BDBD (P2X4 antagonist, 5 µM), assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 8 independent experiments). There was a significantly higher proportion of CD9+ sEVs produced by cells treated with 5-BDBD (*, p <0.05). i Proportion of CD9+, CD63+ and CD81+ sEVs purified from CR7 cells, assessed by Fluo-NTA, normalized to that from CTL cells (n = 5 independent experiments). There was no statistical difference. j Proportion of CD9+-, CD63+- and CD81+-sEVs purified from CTL cells treated with A438079 (P2X7 antagonist, 10 µM), assessed by Fluo-NTA, normalized to that from CTL cells (n = 4 independent experiments). There was no statistical difference. k Intracellular expression of tetraspanins CD9, CD63, CD81 in CTL, CR4 and CR7 cells analysed by flow cytometry. Data are represented in relative MFI with mean ± SD normalized to that in 4T1 cells in CTL condition (n = 8). (**, p < 0.01). NS stands for no statistical difference. l Representative western blot showing the protein content and sEV production in CTL, CR4 and CR7 cells, under both normoxia and hypoxia. Markers of sEVs (CD9, CD63, CD81, TSG101, Flotillin1, Syntinin-1), are enriched in extracellular vesicle fractions, but not GRP94 used as a marker of endoplasmic reticulum which was only identified in total cell protein extract. sEVs produced by CR4 cells demonstrated a higher level of CD9. HSC70 and β-actin were used as control proteins

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Knockdown, Expressing, Concentration Assay, Purification, Produced, Protein Concentration, Zeta Potential Analyzer, Flow Cytometry, Western Blot, Marker, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: EVs from P2X4-deficient cells show a differential expression of proteins involved in endo-lysosome, exosome and autophagosome fusion and maturation. a Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins implicated in late endosome/MVBs transport and fusion to lysosome (GO term reported Supplementary Fig 5b), identified in EVs coming from CTL and CR4 (n=3 independent experiments). *, p < 0.5; **, p < 0.01; *** p < 0.001. b STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CTL cells, thus expressing P2X4, and involved in late endosome/MVBs transport or fusion to lysosome. c Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins involved in early endosome transport, endosome maturation, autophagy and exosome assembly (GO term reported Supplementary Fig 5a), identified in EVs coming from CTL and CR4 (n=3 independent experiments). Statistically different at: *, p < 0.5; **, p < 0.01 and ***, p < 0.001. d STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CR4 cells, thus not expressing P2X4, involved in exosome assembly, autophagy and early endosome transport, endosome maturation. e Representative western blot showing the presence of autophagy markers LC3-II and P62 in CTL, CR4 and CR7 cells, cultivated either under normoxia or hypoxia, and their respective purified EVs

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: sEVs produced from P2X4-expressing cancer cells enhance the invasive capacities of recipient cancer cells. a CTL cancer cells were treated with EVs purified from CTL, CR4 or CR7 cells, grown under hypoxic conditions (1% O2). EVs were stained with green, fluorescent membrane probe PKH67. Representative fluorescent images obtained after 4 h treatment with 20 µg/mL of EVs. Upper, merged images showing DAPI staining for identification of cell nuclei and PKH67 staining showing EVs. Lower, PKH67 staining alone. White arrows indicate EVs incorporated into CTL recipient cells. Scale bars, 75 μm. b Assessment of the uptake as a function of time of PKH67-stained EVs, produced by CTL, CR4, CR7 cells, in CTL cancer recipient cells by flow cytometry (treatment with 20 µg/mL EVs protein). Results indicate a speed of uptake expressed as a ratio of the uptake after 4-h on that after 1-h incubation. Data are represented in rMFI with Mean MFI 4 h normalized to their respective control MFI 1 h, mean ± SD (n = 6 independent experiments). *, indicate a statistical difference at p < 0.05. NS stands for no statistical difference. c Cartoon of the experimental procedure of co-culture assays to assess cell invasiveness performed in hypoxic conditions (1% O2). Invasiveness of recipient CTL was assessed as being their capacity to invade an 8 μm-sized filter covered by a thin layer of Matrigel. These cells were co-cultured with CTL, CR4, CR7 donor cells seeded at the bottom of the well. d Invasive capacities of recipient CTL cells co-cultured with CTL, CR4 or CR7 donor cells, as illustrated in c. Experiments were performed in the absence (vehicle, DMSO) or presence of the nSMAse inhibitor GW4869 (10 µM). Data are normalized to the control condition, in absence of GW4869, and presented as mean ± SD ( n = 6 independent experiments). *, p < 0.05 and **, p < 0.01. NS stands for no statistical difference

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Produced, Expressing, Purification, Staining, Membrane, Flow Cytometry, Incubation, Control, Co-Culture Assay, Cell Culture

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: Differential roles of P2X4 and P2X7 receptors in inducing sEV production under hypoxic conditions. CTL, CR4 (knock-down for the expression of P2X4) and CR7 (knock-down for the expression of P2X7) cells were grown under hypoxic conditions (1% O2) for a duration of 48 h prior to conducting analyses. a Size distribution and concentration of EVs purified from CTL, CR4 or CR7 cells grown under hypoxia, assessed by NTA (n = 12 independent experiments for CTL and CR4, n = 6 independent experiments for CR7). b Relative concentrations of sEVs per mL of supernatants, expressed relatively to that for CTL, in the same conditions than in a. The concentration of sEV produced by CR4 cells was significantly higher as compared to CTL (**, p <0.01). NS stands for not statistically different. c Protein concentrations of sEV extracts from same conditions than in a. There was a significantly higher protein concentration of sEVs extracts from CR4 cells, as compared to CTL (*, p <0.05) or to CR7 (*, p <0.05) cells. NS stands for not statistically different. d Relative concentration of sEVs per mL of supernatants coming from CTL cells treated with either 5-BDBD (P2X4 antagonist, 5µM) or A438079 (P2X7 antagonist, 10 µM) and expressed relatively to the CTL condition (vehicle). There was a significantly higher concentration of sEV produced by cells submitted to the P2X4 antagonist as compared to CTL (*, p <0.05) but not with cells treated with the P2X7 antagonist. NS stands for not statistically different. e Mean size (in nm) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in same conditions than in a. EVs produced by CR4 cells were significantly smaller than CTL (*, p <0.05). NS stands for not statistically different. f Zeta potential (in mV) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in similar conditions than in a. There was no difference between the three groups (n = 4 independent experiments). g Proportion of CD9+, CD63+ and CD81+ sEV purified from CR4 cells supernatants, assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 12 independent experiments). There was a significantly higher proportion of CD9+ sEV produced by CR4 cells compared to CTL cells (*, p <0.05). h Proportion of CD9+, CD63+ and CD81+ sEVs purified from supernatants of CTL cells treated with 5-BDBD (P2X4 antagonist, 5 µM), assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 8 independent experiments). There was a significantly higher proportion of CD9+ sEVs produced by cells treated with 5-BDBD (*, p <0.05). i Proportion of CD9+, CD63+ and CD81+ sEVs purified from CR7 cells, assessed by Fluo-NTA, normalized to that from CTL cells (n = 5 independent experiments). There was no statistical difference. j Proportion of CD9+-, CD63+- and CD81+-sEVs purified from CTL cells treated with A438079 (P2X7 antagonist, 10 µM), assessed by Fluo-NTA, normalized to that from CTL cells (n = 4 independent experiments). There was no statistical difference. k Intracellular expression of tetraspanins CD9, CD63, CD81 in CTL, CR4 and CR7 cells analysed by flow cytometry. Data are represented in relative MFI with mean ± SD normalized to that in 4T1 cells in CTL condition (n = 8). (**, p < 0.01). NS stands for no statistical difference. l Representative western blot showing the protein content and sEV production in CTL, CR4 and CR7 cells, under both normoxia and hypoxia. Markers of sEVs (CD9, CD63, CD81, TSG101, Flotillin1, Syntinin-1), are enriched in extracellular vesicle fractions, but not GRP94 used as a marker of endoplasmic reticulum which was only identified in total cell protein extract. sEVs produced by CR4 cells demonstrated a higher level of CD9. HSC70 and β-actin were used as control proteins

Article Snippet: The P2X4 antagonist 5-BDBD and the P2X7 antagonist A438079 were both purchased from Tocris (Bio-Techne, France).

Techniques: Knockdown, Expressing, Concentration Assay, Purification, Produced, Protein Concentration, Zeta Potential Analyzer, Flow Cytometry, Western Blot, Marker, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: EVs from P2X4-deficient cells show a differential expression of proteins involved in endo-lysosome, exosome and autophagosome fusion and maturation. a Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins implicated in late endosome/MVBs transport and fusion to lysosome (GO term reported Supplementary Fig 5b), identified in EVs coming from CTL and CR4 (n=3 independent experiments). *, p < 0.5; **, p < 0.01; *** p < 0.001. b STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CTL cells, thus expressing P2X4, and involved in late endosome/MVBs transport or fusion to lysosome. c Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins involved in early endosome transport, endosome maturation, autophagy and exosome assembly (GO term reported Supplementary Fig 5a), identified in EVs coming from CTL and CR4 (n=3 independent experiments). Statistically different at: *, p < 0.5; **, p < 0.01 and ***, p < 0.001. d STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CR4 cells, thus not expressing P2X4, involved in exosome assembly, autophagy and early endosome transport, endosome maturation. e Representative western blot showing the presence of autophagy markers LC3-II and P62 in CTL, CR4 and CR7 cells, cultivated either under normoxia or hypoxia, and their respective purified EVs

Article Snippet: The P2X4 antagonist 5-BDBD and the P2X7 antagonist A438079 were both purchased from Tocris (Bio-Techne, France).

Techniques: Quantitative Proteomics, Expressing, Western Blot, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: sEVs produced from P2X4-expressing cancer cells enhance the invasive capacities of recipient cancer cells. a CTL cancer cells were treated with EVs purified from CTL, CR4 or CR7 cells, grown under hypoxic conditions (1% O2). EVs were stained with green, fluorescent membrane probe PKH67. Representative fluorescent images obtained after 4 h treatment with 20 µg/mL of EVs. Upper, merged images showing DAPI staining for identification of cell nuclei and PKH67 staining showing EVs. Lower, PKH67 staining alone. White arrows indicate EVs incorporated into CTL recipient cells. Scale bars, 75 μm. b Assessment of the uptake as a function of time of PKH67-stained EVs, produced by CTL, CR4, CR7 cells, in CTL cancer recipient cells by flow cytometry (treatment with 20 µg/mL EVs protein). Results indicate a speed of uptake expressed as a ratio of the uptake after 4-h on that after 1-h incubation. Data are represented in rMFI with Mean MFI 4 h normalized to their respective control MFI 1 h, mean ± SD (n = 6 independent experiments). *, indicate a statistical difference at p < 0.05. NS stands for no statistical difference. c Cartoon of the experimental procedure of co-culture assays to assess cell invasiveness performed in hypoxic conditions (1% O2). Invasiveness of recipient CTL was assessed as being their capacity to invade an 8 μm-sized filter covered by a thin layer of Matrigel. These cells were co-cultured with CTL, CR4, CR7 donor cells seeded at the bottom of the well. d Invasive capacities of recipient CTL cells co-cultured with CTL, CR4 or CR7 donor cells, as illustrated in c. Experiments were performed in the absence (vehicle, DMSO) or presence of the nSMAse inhibitor GW4869 (10 µM). Data are normalized to the control condition, in absence of GW4869, and presented as mean ± SD ( n = 6 independent experiments). *, p < 0.05 and **, p < 0.01. NS stands for no statistical difference

Article Snippet: The P2X4 antagonist 5-BDBD and the P2X7 antagonist A438079 were both purchased from Tocris (Bio-Techne, France).

Techniques: Produced, Expressing, Purification, Staining, Membrane, Flow Cytometry, Incubation, Control, Co-Culture Assay, Cell Culture