Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: Piezo1 is highly expressed in pancreatic cancer cells and tissues (A and B) Piezo1 was highly expressed in the BxPC3 pancreatic cancer cell line relative to that in the other two pancreatic cancer cell lines and normal pancreatic ductal cells. Values analyzed from experiments performed at least in triplicate and presented as means ± SEM. ∗∗∗p<0.001, one-way ANOVA test. (C and D) Immunohistochemistry showed Piezo1 being abundantly expressed in the pancreatic ductal cells of patients with PDAC relative to that in paired adjacent NP tissues. PC: pancreatic cancer, NP: noncancerous pancreatic. Scale bars: 50 or 25 μm. Black arrows denoted the Piezo1 expression. Values analyzed from experiments performed at least in triplicate and presented as means ± SEM. ∗∗∗∗p<0.0001, Student's t -test. (E) Piezo1 was mainly distributed in the BxPC3 cell membrane, co-localized with the membrane maker WGA as determined by immunofluorescence. Scale bars: 20 μm.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Immunohistochemistry, Expressing, Membrane, Immunofluorescence

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: High expression of Piezo1 is associated with poor survival and patients with advanced clinical stage of pancreatic cancer (A–C) Oncomine, GEO, and GEPIA datasets show that Piezo1 expression was increased in patients with PDAC or PAAD (Oncomine, n = 8 for PDAC vs . n = 6 for normal; GEO, n = 39 for PDAC vs . n = 39 for normal; GEPIA, n = 179 for PAAD vs . n = 171 for normal). Data are represented as mean ± SEM. ∗p<0.05, ∗∗∗∗p<0.0001, Student's t -test. (D–G) GEPIA database and KM plotter showed no significant difference in OS between the high- and low-Piezo1 expression groups (p = 0.37 in GEPIA, p = 0.11 in KM plotter); high Piezo1 expression was closely related to low DFS in pancreatic cancer cases (p = 0.034 in GEPIA, p = 0.0016 in KM plotter). (H) Piezo1 expression was higher in patients with clinical Stage II than those with Stage I (p = 0.0063; Stage I, n = 21; Stage II, n = 144; Stage III, n = 4; Stage IV, n = 4). Data are represented as mean ± SEM. ∗p<0.05, one-way ANOVA test. (I) Piezo1 expression was higher in patients with grade 3 tumor than those with grades 1 and 2 tumor; however, no significant difference was found between these groups.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Expressing

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: Piezo1 contributes to apoptosis of pancreatic cancer cells induced by ultrasound with microbubbles (A and B) GsMTx4 alleviated the apoptosis of pancreatic cancer cells induced by US with MBs after incubation for 24 h or 48 h. (C and D) Partial blockage of Piezo1 by siRNA also alleviated the apoptosis of pancreatic cancer cells when exposed to US with MBs. (E–H) Western blot analysis showed that the expression of Cyto-C and Bax was increased, whereas that of Bcl-2 was decreased after GsMTx4 or siPiezo1 usage in both 24 h and 48 h groups. All experiments were conducted in triplicate, with the results presented as means ± SEM. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, two-way ANOVA test.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Incubation, Western Blot, Expressing

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: Piezo1 contributes to mitochondrial dysfunction of pancreatic cancer cells induced by ultrasound with microbubbles (A) The JC-1 dyes exhibiting green and red fluorescence represented the monomers and aggregates of the JC-1, respectively. The JC-1 monomers increased during mitochondrial dysfunction. Both GsMTx4 and siPiezo1 alleviated the production of the JC-1 monomers of pancreatic cancer cells when exposed to US with MBs. Scale bars: 100 μm. (B, C, E, and F) Flow cytometry showed that the percentage of the JC-1 monomers was increased in the US with MBs group and was alleviated by GsMTx4 or siPiezo1 in both the 24 h and 48 h groups. P2 indicated the percentage of the JC-1 dye exhibiting green fluorescence. (D and G) ATP production was decreased in the US + MBs group, and GsMTx4 or siPiezo1 partly alleviated this condition for pancreatic cancer cells. All experiments were run in triplicate, with the results presented as means ± SEM. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, two-way ANOVA test.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Fluorescence, Flow Cytometry

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: Piezo1 partially mediates influx of Ca 2+ induced by ultrasound with microbubbles in pancreatic cancer cells (A) Fluo-8 AM is an indicator of Ca 2+ . Ca 2+ influx occurred when the cells were exposed to US with MBs. The use of GsMTx4 or siPiezo1 partly decreased Ca 2+ influx, but the reduction was more significant in the US + MBs + siPiezo1 group. Scale bar: 50 μm. Hoechst staining was conducted to allow for the fluorescent staining of the cell nucleus. (B) Cytosolic Ca 2+ immunofluorescence was measured by flow cytometry and showed similar results.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Staining, Immunofluorescence, Flow Cytometry

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet: Knockdown of Piezo1 reduces the therapeutic effect of ultrasound with microbubbles in vivo (A and B) Tumor volume and body weight were measured before each treatment conducted every other day. Day 1 represented the first treatment. The US + MBs + Lv-NC group showed the smallest increase in tumor volume. (C) Representative images of the mouse in each group on days 1, 7, and 13. H&E staining and TUNEL staining showed the extent of cell apoptosis in each group. Scale bars: 20 and 25 μm. Data are presented as the means ± SEM. ∗∗p<0.01, ∗∗∗∗p<0.0001, two-way ANOVA test.

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Knockdown, In Vivo, Staining, TUNEL Assay

Journal: iScience

Article Title: Mechanosensitive channel Piezo1 induces cell apoptosis in pancreatic cancer by ultrasound with microbubbles

doi: 10.1016/j.isci.2022.103733

Figure Lengend Snippet:

Article Snippet: The cells were incubated with an anti-Piezo1 antibody (rabbit, 1:400, Proteintech Cat# 15939-1-AP, RRID: AB_2231460 , Chicago, USA) overnight at 4°C followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature.

Techniques: Recombinant, Avidin-Biotin Assay, Plasmid Preparation, Staining, TUNEL Assay, Software, Flow Cytometry

Journal: Nanomaterials

Article Title: The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au 25 Probes

doi: 10.3390/nano12060923

Figure Lengend Snippet: Fluorescence imaging of BSA-biotin-fitc labeled HER-2 in SK cells by avidin and primary antibody-biotin at different time points.

Article Snippet: Human HER-2 antibody, HER-2 biotinylated antibody, Avidin and trastuzumab were obtained from Abcam.

Techniques: Fluorescence, Imaging, Labeling, Avidin-Biotin Assay

Journal: Nanomaterials

Article Title: The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au 25 Probes

doi: 10.3390/nano12060923

Figure Lengend Snippet: ( a ) Fluorescence imaging of 12 μM BSA-biotin-Au 25 labeled MDA-MB-231 cells at different time points. ( b ) HER-2 protein expressed in MDA-MB-231 cells before and after paclitaxel stimulation ( c ) Fluorescence imaging of BSA-biotin-Au 25 labeled MDA-MB-231 cells before and after paclitaxel stimulation.

Article Snippet: Human HER-2 antibody, HER-2 biotinylated antibody, Avidin and trastuzumab were obtained from Abcam.

Techniques: Fluorescence, Imaging, Labeling