Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Mass spectrometry data showing proteins enriched in the plasma membrane (PM) > fivefold (fold changes represented using triangle plots; OC/ND ratio on the right axis) when cells are under overconfluence (OC) (red bars) relative to normal density (ND) conditions (blue bars). Ion channels are marked with red boxes, where TRPV4 shows about a 160-fold increased association with the plasma membrane under OC conditions. ( B ) Proteins near and on the plasma membrane were pulled down after cell surface biotinylation with streptavidin beads and immunoblotted for TRPV4. TRPV4 is significantly associated with the plasma membrane in OC MCF10DCIS.com cells. In MCF10CA1a cells, TRPV4 appears to be associated with the plasma membrane under both ND and OC conditions, with a slight increase under OC conditions. ( C ) Immunoblots of whole-protein lysates demonstrate similar overall TRPV4 protein levels across MCF10A cell derivatives, regardless of cell density. This indicates that the differing plasma membrane association of TRPV4 is due to trafficking changes, not expression level changes. GAPDH is used as a loading control. ( D ) Representative immunofluorescence (IF) images by confocal microscopy show TRPV4 (red) localization compared to the control protein transferrin receptor (TfR; green) in MCF10A, MCF10AT1, MCF10DCIS.com, and MCF10CA1a cells under ND and OC conditions. DAPI (blue) staining was used for visualizing the nuclei. As observed in the biochemical data in ( B-C ), cell crowding induces the relocation of TRPV4 to the plasma membrane in MCF10DCIS.com cells. TRPV4 is associated with the plasma membrane in ND MCF10CA1a cells, with a clear elevated association in OC cells. Scale bar = 10 μm. ( E ) Plasma membrane-associated TRPV4 (%) is quantified for the four cell lines under ND and OC conditions by line analysis, showing a significant increase in both MCF10DCIS.com cells and MCF10CA1a cells due to cell crowding. The number of cells used for line analyses (technical replicates merged from three independent experimental repeats) was as follows: MCF10A (ND: 6; OC: 12), MCF10AT1 (ND: 6; OC: 11), MCF10DCIS.com (ND: 12; OC: 8), and MCF10CA1a (ND: 10; OC: 10). ( F ) IF images show that hyperosmotic conditions induced by PEG 300 (74.4 mOsm/Kg) treatment also relocate TRPV4 (red) to the plasma membrane in MCF10DCIS.com cells. TfR localization remains consistent under hyperosmotic conditions. Increased relocation is also observed in MCF10CA1a cells. Scale bar = 10 μm. ( G ) The increased plasma membrane association of TRPV4 due to hyperosmotic stress is quantified by line analysis. The number of cells used for line analyses (technical replicates merged from two independent experimental repeats) was as follows: MCF10A (ND control: 6; ND +4% PEG: 15), MCF10AT1 (ND control: 6; ND +4% PEG: 9), MCF10DCIS.com (ND control: 12; ND +4% PEG: 8), and MCF10CA1a (ND control: 10; ND +4% PEG: 21). Scale bar = 10 μm. ( H ) Representative regions of interest (ROIs) of TRPV4-stained immunohistochemistry (IHC) images in different pathology phenotypes. High-grade ductal carcinoma in situ (DCIS) and invasive ductal cancer (IDC) ROIs clearly exhibit plasma membrane association of TRPV4. Two high-grade DCIS IHC images were acquired by two different people and both show plasma membrane-associated TRPV4. Scale bar = 20 μm. ( I ) Statistical results from independent histological evaluations of pathologies and TRPV4 distributions of 97 ROIs from 39 patient specimens indicate a high correlation (>70%) of plasma membrane association of TRPV4 with high-grade DCIS or IDC pathologies. Y/N: Yes/no, indicating both pathologists agreed that PM ion channels were present/absent. E: Equivocal, indicating the pathologists disagreed. Significantly high proportions of high-grade DCIS (75%) and IDC (73%) ROIs exhibited plasma membrane TRPV4 association, which was not observed in lower-risk cases. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: p>0.05. Figure 3—source data 1. Original data corresponding to panel A. Figure 3—source data 2. Original data corresponding to panel B. Figure 3—source data 3. Original data corresponding to panel B. Figure 3—source data 4. Original data corresponding to panel C. Figure 3—source data 5. Original data corresponding to panel C. Figure 3—source data 6. Original data corresponding to panels E and G. Figure 3—source data 7. Gene and protein names that showed more than a fivefold increase in plasma membrane association under overconfluence (OC) conditions relative to normal density (ND) conditions in MCF10DCIS.com cells were identified by mass spectrometry. Ion channels among these are highlighted in bold. Figure 3—source data 8. Top 25 genes and corresponding proteins that exhibited more than a 100-fold increase in plasma membrane association under OC conditions compared to ND conditions in MCF10A cells (left), and more than a fivefold increase in MCF10AT1 (middle) and MCF10CA1a (right) cells, as identified by mass spectrometry. One ion transporter showing plasma membrane relocation under OC conditions in MCF10CA1a cells is highlighted in bold.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Mass Spectrometry, Membrane, Western Blot, Expressing, Control, Immunofluorescence, Confocal Microscopy, Staining, Immunohistochemistry, In Situ

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: Immunofluorescence images ( A ) and immunoblots ( B ) verified the binding specificity of TRPV4 antibodies in control normal density (ND) DCIS.com cells and TRPV4-depleted cells treated with either 2 μM siRNA (Dharmacon On Target Plus SMART Pool L-004195-00-0005) or 1 and 2 μg shRNA (shRNA pool, Santa Cruz sc-61726-SH) for 36 hr. ( A ) Compared to invariant transferrin receptor (TfR) staining (green), TRPV4 (red) depletion was 40% with 1 µg shRNA and 80% with 2 µg shRNA, as quantified by intensity measurements. DAPI (blue) is also shown in the merged images. All images were visualized using the same intensity settings. Scale bar = 20 µm. ( B ) Immunoblot results confirmed this dose-responsive depletion of TRPV4, with 33% reduction observed at 1 µg shRNA and 51% at 2 µg. Figure 3—figure supplement 1—source data 1. Original data for panel B. Figure 3—figure supplement 1—source data 2. Raw gel image for the immunoblot data.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Immunofluorescence, Western Blot, Binding Assay, Control, shRNA, Staining

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) We used the plasma membrane marker DiIC18(3) to confirm the association of TRPV4 with the plasma membrane in MCF10DCIS.com and MCF10CA1a cells under overconfluence (OC) conditions. As described in the Methods section, we stained DiIC18(3) in live cells and co-stained TRPV4 and DAPI in fixed and permeabilized cells. The immunofluorescence (IF) images show TRPV4 (red), DiIC18(3) (DiI, green), and DAPI (blue). The line profile plots on the right demonstrate colocalization of TRPV4 with DiIC18(3) at the plasma membrane (PM), marked by the green DiIC18(3) signal, which overlaps with the red TRPV4 signal. The nucleus location (NUC) is indicated by the blue DAPI signal. Scale bar = 20 μm. ( B ) We examined the relocation of KCNN4 and PIEZO1 to the plasma membrane in response to cell crowding. Mass spectrometry showed a slight increase in KCNN4 at the plasma membrane under OC conditions. In ND MCF10DCIS.com cells, KCNN4 was predominantly cytosolic, whereas PIEZO1 showed some plasma membrane association. Under OC conditions, both KCNN4 and PIEZO1 showed a modest relocation to the plasma membrane. ( C ) Line analysis confirmed a slight increase in plasma membrane association for both KCNN4 and PIEZO1 under OC conditions compared to ND conditions. Scale bar = 20 μm. For the statistical analysis, we employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value. The levels of statistical significance are denoted as follows: **** indicates p<0.0001, *** indicates p<0.001, * indicates p<0.1, and ‘ns’ indicates p>0.05. Figure 3—figure supplement 2—source data 1. Original data corresponding to panel A. Figure 3—figure supplement 2—source data 2. Original data corresponding to panel C. Figure 3—figure supplement 2—source data 3. Original data corresponding to panel C.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Membrane, Marker, Staining, Immunofluorescence, Mass Spectrometry, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Relative TRPV4 associations with the plasma membrane (PM), cytoplasm (Cyt), and nucleus (Nuc) are plotted for ND versus OC conditions in MCF10A (10 A), MCF10AT1 (10AT1), MCF10DCIS.com (10DCIS.com), and MCF10CA1a (10CA1a) cells. ( B ) Similar analyses were performed to compare the intracellular TRPV4 associations in PM, Cyt, and Nuc between control ND and 74.4 or 148.8 mOsm/kg PEG 300 treatment groups. We employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value for the statistical analysis. The levels of statistical significance are denoted as follows: **** indicates p<0.0001, *** indicates p<0.001, * indicates p<0.1, and ‘ns’ indicates p>0.05. Figure 3—figure supplement 3—source data 1. Original data corresponding to panel A and B.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Membrane, Control, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) To determine whether the seemingly plasma membrane-associated TRPV4 under hyperosmotic conditions was indeed localized at the plasma membrane of live cells, we used an extracellular domain (ECD) TRPV4 antibody. See the Methods section for sample preparation details. Live-cell imaging with 60 X confocal microscopy revealed distinct binding of the ECD TRPV4 antibodies (red) exclusively in MCF10DCIS.com cells treated with 74.4 mOsm/kg PEG 300 for 15 min. No binding was observed in the untreated control group. Brightfield images are shown below the fluorescence images. ( B ) Line analysis demonstrated that ~60% of TRPV4 was plasma membrane-associated under 2% (74.4 mOsm/kg) PEG 300 conditions, compared to ~0% in untreated cells. Statistical analysis was conducted using a nonparametric Mann-Whitney test with two-tailed p-values. Statistical significance levels are indicated as follows: ****p<0.0001, ***p<0.001, *p<0.1, and ‘ns’ denotes p>0.05. Figure 3—figure supplement 4—source data 1. Original data corresponding to panel B.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Membrane, Sample Prep, Live Cell Imaging, Confocal Microscopy, Binding Assay, Control, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Previously overconfluence (OC) MCF10DCIS.com cells were trypsinized and reseeded under normal density (ND) conditions. These cells were fixed and stained for TRPV4 (red) and DAPI (blue) for immunofluorescence (IF) imaging. While TRPV4 was elevated at the plasma membrane under OC conditions, it redistributed to the cytoplasm after the cells were reseeded under ND conditions. ( B ) The reseeded ND cells responded similarly to hyperosmotic stress (74.4 mOsm/Kg PEG 300 for 15 min), with TRPV4 relocating to the plasma membrane. ( C ) Line analysis demonstrated that the reseeded ND cells exhibited a TRPV4 distribution pattern comparable to their initial ND counterparts without (blue circles) and with (red circles) hyperosmotic conditions. Statistical analysis was performed using a nonparametric Mann-Whitney test with two-tailed p-values. Statistical significance levels are denoted as follows: ****p<0.0001, ***p<0.001, *p<0.1, and ‘ns’ indicates p>0.05. Figure 3—figure supplement 5—source data 1. Original data corresponding to panel C.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Staining, Immunofluorescence, Imaging, Membrane, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Confluent cell density (Con) also triggered the relocation of TRPV4 (red) to the plasma membrane, similar to OC conditions, as shown in immunofluorescence (IF) image (left). We used DiIC18(3) to indicate the plasma membrane location (green; DiI; middle image), and the merged image (right) of TRPV4 and DiIC18(3) shows excellent overlay at the plasma membrane (PM), as illustrated in the line profile plots from our line analysis. DAPI (blue) staining was used to locate the nucleus (NUC). Scale bar = 20 μm. ( B ) Confluent cell density resulted in lower intracellular calcium levels compared to less confluent cells. Live MCF10DCIS.com cells stained with Fluo-4 were imaged using confocal microscopy at 488 nm. Two line profiles crossing peripheral cells (less confluent than confluent cells) and adjacent confluent cells within the clusters clearly showed that the peripheral cells have a higher Fluo-4 signal (700 au) compared to the confluent cells within the cluster (200 au), highlighting the crowding-induced intracellular calcium reduction. Background regions (bg) were noted with cyan dashed-lines in the fluorescent image and in the plots. Scale bar = 100 μm. Figure 4—figure supplement 1—source data 1. Original data corresponding to panel A. Figure 4—figure supplement 1—source data 2. Original data corresponding to panel B.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Membrane, Immunofluorescence, Staining, Confocal Microscopy

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) To compare intracellular calcium (Ca² + ) levels, we used a Fluo-4 AM assay, where green fluorescence intensity increases with higher intracellular Ca² + levels. Calcium levels are significantly lower in confluent (Con) MCF10DCIS.com cells. ( B ) The temporal progression of averaged Fluo-4 intensity in normal density (ND) MCF10DCIS.com cells (blue curve) in the box shown on the left image is compared with that of Con cells (red curve). Fluo-4 intensity is consistently lower in Con cells than in ND cells for approximately 25 min (200 ms acquisition time and 30 s time interval). ( C ) Fluo-4 intensity reduction due to cell crowding is significant in MCF10DCIS.com cells. 10 images were used for calculating average Fluo-4 intensities for both ND and Con cells. ( D - H ) Pharmacological inhibition of TRPV4 with 1 nM GSK219 generates dips in the Fluo-4 signal. Fluo-4 images at the baseline ( t 1 ) and the dip ( t 2 ) post 1 nM GSK219 are compared in MCF10DCIS.com cells between ND ( D ) and Con ( F ) conditions. The Fluo-4 intensity time traces are compared between ND (blue; E) and Con (red; G) conditions, showing that the magnitude of the dip (marked as ΔCa) is significantly lower in Con cells, where TRPV4 activity is largely inhibited under cell crowding conditions. Notably, the magnitude of ΔCa increased with higher GSK219 doses (1 nM vs. 0.2 nM), but remained significantly lower in Con MCF10DCIS.com cells, with smaller changes observed under the 0.2 nM GSK219 condition. The number of ΔCa measurements ( H ) (technical replicates merged from two independent experimental repeats) was as follows: MCF10DCIS.com (ND+ GSK219 Low: 9; ND+ GSK219 High: 12; Con+ GSK219 High: 10; Con+ GSK219 Low: 14). ( I - M ) TRPV4 activation with 0.2 pM GSK101 leads to a small spike in ND cells ( I, J ). However, in Con cells, the same GSK101 treatment leads to a notably larger spike in Fluo-4 intensity, indicating that TRPV4 inhibition and subsequent relocation to the plasma membrane by cell crowding primes the ion channels for activation. GSK101 treatment also leads to a dose-dependent increase in the spike magnitude with a higher GSK101 concentration being strikingly high in Con MCF10DCIS.com cells (0.05 pM: 101 L; 0.2 pM: 101 H). The number of ΔCa measurements ( M ) (technical replicates merged from two independent experimental repeats) was as follows: MCF10DCIS.com (ND+ GSK101 Low: 7; ND+ GSK101 High: 9; Con+ GSK101 Low: 9; Con+ GSK101 High: 9). ( N-Q ) TRPV4 activation status-dependent intracellular localization changes. ( N ) IF images of TRPV4 (red) and TfR (green) in ND MCF10DCIS.com cells show that GSK101 does not increase plasma membrane association of TRPV4. However, GSK219 significantly relocates TRPV4 to the plasma membrane in a dose-dependent manner ( for all dose cases), similar to ND cells treated with 74.4 mOsm/Kg PEG 300. ( O ) In OC cells, while GSK219 does not significantly alter TRPV4 association with the plasma membrane, GSK101 depletes plasma membrane TRPV4 in a dose-dependent manner ( for all dose cases), suggesting that TRPV4 activation status affects its trafficking. Relative plasma membrane associations with different treatments are quantified for ND ( P ) and OC ( Q ) cells using line analysis. The number of line analyses (P, Q) (technical replicates merged from two independent experimental repeats) was as follows: ND and OC MCF10DCIS.com (control: 12 and 8; GSK219 Low: 6 and 7; GSK219 High: 6 and 13; GSK101 Low: 7 and 8; GSK101 High: 7 and 10; 2% PEG: 12 and 12). ( R ) The values of Fluo-4 spikes by GSK101 and dips by GSK219 show a linear relationship ( R ²~0.69) with the plasma membrane TRPV4 association, indicating a negative correlation between them. This reinforces the observation that TRPV4 inhibition increases its association with the plasma membrane, while activation shows the reverse effect. ( S-V ) Compared to the Fluo-4 intensity in control MCF10DCIS.com cells (S, T), shRNA showed similar baseline Fluo-4 levels ( U, V ). However, hyperosmotic stress by 74.4 mOsm/Kg PEG 300 (light gray box) led to a noticeable spike only in control ND cells. Additionally, cell crowding conditions (Con) led to a decreased Fluo-4 level (at t 1 baseline in the image in S and red time trace in T); but a reduced in Fluo-4 level difference in shRNA-treated MCF10DCIS.com cells ( t 1; U) compared to control cases ( S, T ), as shown in the image and time trace ( t 1; V). ( W ) Relative Fluo-4 time-averaged intensities are plotted for individual control ND (blue) vs. Con (red) cells, and shRNA-treated ND (semi-transparent blue) vs. Con (semi-transparent red) cells. Intracellular calcium levels in shRNA ND cells are lower than those in control ND cells, reflecting the reduced number of TRPV4 channels. The decrease in calcium levels by crowding (Con) in shRNA cells is clearly lower than in control cells, reflecting the importance of TRPV4 in mechanosensing cell volume reduction. The number of Fluo-4 average measurements (technical replicates merged from two independent experimental repeats) was as follows: MCF10DCIS.com control and TRPV4 shRNA groups (ND: 19 and 18; Con: 11 and 17). ( X ) PEG 300-induced calcium spikes are significantly lower in shRNA cells (semi-transparent gray) than in control cells (gray), reinforcing TRPV4’s crucial role in MCF10DCIS.com mechanotransduction. The number of ΔCa measurements under 2% PEG 300 condition was as follows: ND MCF10DCIS.com (ND: 19; TRPV4shRNA: 16). ( Y ) TRPV4 silencing significantly reduced the mechanosensing cell volume reduction effect. Control ND cells underwent a 48% volume reduction in response to 74.4 mOsm/Kg PEG 300, whereas TRPV4-silenced cells reduced their volume by only 27%. The number of single-cell volume measurements (technical replicates merged from two independent experimental repeats) was as follows: ND and TRPV4shRNA treated MCF10DCIS.com (Control: 11 and 13; 2% PEG 8 and 11). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: p>0.05. Figure 4—source data 1. Original data corresponding to panel B. Figure 4—source data 2. Original data corresponding to panel C. Figure 4—source data 3. Original data corresponding to panel E. Figure 4—source data 4. Original data corresponding to panel G. Figure 4—source data 5. Original data corresponding to panel H. Figure 4—source data 6. Original data corresponding to panel J. Figure 4—source data 7. Original data corresponding to panel L. Figure 4—source data 8. Original data corresponding to panel M. Figure 4—source data 9. Original data corresponding to panel P. Figure 4—source data 10. Original data corresponding to panel Q. Figure 4—source data 11. Original data corresponding to panel R. Figure 4—source data 12. Original data corresponding to panel T. Figure 4—source data 13. Original data corresponding to panel V. Figure 4—source data 14. Original data corresponding to panel W. Figure 4—source data 15. Original data corresponding to panel X. Figure 4—source data 16. Original data corresponding to panel Y.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Fluorescence, Inhibition, Activity Assay, Activation Assay, Membrane, Concentration Assay, Control, shRNA

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Cell viability assays in which viable cells were counted based on trypan blue exclusion after 2 d of GSK101 or GSK219 treatment in the specified concentration ranges of ductal carcinoma in situ (DCIS) in normal density (ND) (white bars) and overconfluence (OC) (gray bars) conditions. Treatment ranges were selected so that cell viability was >90%. Concentrations used for dose-dependent assays were 0.05 and 0.2 pM for GSK101, and 0.2 and 1 nM for GSK219 (marked by dotted red boxes). ( B ) Representative confocal microscopy immunofluorescence images showed effects of GSK101 (0.05 and 0.2 pM) or GSK219 (0.2 and 1 nM) treatment for two days on TRPV4 (red) and control transferrin receptor (TfR; green) distributions in ND and OC cells in a dose-dependent manner. DAPI (blue) signal is shown in the merged images. Scale bar = 20 μm. Figure 4—figure supplement 2—source data 1. Original data corresponding to panel A.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Concentration Assay, In Situ, Confocal Microscopy, Immunofluorescence, Control

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) Using the Fluo-4 assay, we observed an initial calcium spike (marked as ‘Rise’) in ND MCF10DCIS.com cells in response to 74.4 mOsmol/kg PEG 300, due to osmotic water outflow. This was followed by a homeostatic relaxation, aimed at restoring calcium levels, which likely involved the inhibition of ion channels like TRPV4, leading to their plasma membrane relocation. Scale bars = 20 μm. ( B ) The same hyperosmotic condition (74.4 mOsm/Kg PEG 300 for 15 min) led to the relocation of KCNN4 and PIEZO1 to the plasma membrane, similar to the relocations observed under OC conditions. Line analysis results showed the relative relocations of each channel in response to hyperosmotic (PEG) and cell crowding (OC) stresses, compared to ND conditions. Figure 4—figure supplement 3—source data 1. Original data corresponding to panel A. Figure 4—figure supplement 3—source data 2. Original data corresponding to panel B.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Inhibition, Membrane

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A-C ). MCF10DCIS.com cell volume changes with TRPV4 inhibition and activation. ( A ) In ND MCF10DCIS.com cells, TRPV4 agonist GSK101, which did not alter plasma membrane association of TRPV4, did not affect cell volume. Conversely, TRPV4 inhibitor GSK219, which increased plasma membrane association in a dose-dependent manner, reduced cell volume, with the effect of 1 nM GSK219 (219 H) being similar to that of 74.4 mOsm/Kg (2%) PEG 300. ( B ) Under OC conditions, GSK101, which led to significant Fluo-4 spikes, increased cell volume in a dose-dependent manner, while GSK219 and PEG only mildly reduced cell volume. ( C ) Cell volume changes in MCF10DCIS.com cells show an inverse relationship ( R ²=0.59) with plasma membrane association of TRPV4, reflecting the activation status of the channel. The number of single-cell volume measurements (technical replicates merged from three independent experimental repeats): ND ( A ) and OC ( B ) MCF10DCIS.com cells (Control: 33 and 43; GSK101 0.05 pM: 19 and 15; GSK101 0.2 pM: 9 and 22; GSK219 0.1 nM: 10 and 36; GSK101 1 nM: 15 and 9; 2% PEG 300: 23 and 8). ( D-F ) Cell invasiveness increases with greater cell volume reduction and plasma membrane association of TRPV4. ( D ) Cell invasiveness significantly increased with higher GSK219 concentrations under ND conditions. ( E ) GSK101 under OC conditions caused a notable decrease in cell invasiveness in a dose-dependent manner. ( F-G ) Plasma membrane association of TRPV4 predictably reports cell invasiveness (R²~0.69; F), while cell invasiveness and cell volume are inversely related (R²~0.69; G), reinforcing our observation that cell volume reduction promotes cell invasiveness. The number of invasive cell fraction measurements (technical replicates merged from two independent experimental repeats): ND (D) and OC ( E ) MCF10DCIS.com cells (Control: 6 and 4; GSK101 0.05 pM: 4 and 4; GSK101 0.2 pM: 4 and 4; GSK219 0.1 nM: 4 and 4; GSK101 1 nM: 4 and 4; 2% PEG 300: 4 and 7). ( H-M ) To assess if cell motility also follows the trend of cell invasiveness, we performed a single-cell motility assay by tracking nuclear WGA in individual live cells every 60 s for 25 min. ( H ) Representative trajectories of individual cells were color-coded to reflect displacement at each time interval. Compared to untreated ND cells, 0.2 pM GSK101 treatment slowed overall cell diffusion, while 1 nM GSK219 and 74.4 mOsm/Kg PEG 300 treatments increased cell diffusion. ShRNA TRPV4 (Sh-ctrl) increased cell motility under ND conditions. However, with TRPV4 depletion, treatment with 74.4 mOsm/Kg PEG 300 failed to increase cell diffusivity ( D ) in shRNA-treated cells (Sh-PEG), unlike in the untreated cells. Scale bar = 200 μm. Using single-cell analysis, we quantified cell diffusivity ( D ) and speed ( v ; movement directionality). ( I ) GSK101 treatment significantly reduced D , while GSK219 and PEG 300 notably increased it. shRNA TRPV4 also increased ND cell D , but PEG treatment did not change D in the shRNA-treated cells. ( J ) GSK101, GSK219, PEG 300, and shRNA treatments increased v, with GSK101 causing the most significant increase. The directionality of shRNA-treated ND cells was unaffected by PEG treatment. The number of single-cell motility measurements of MCF10DCIS.com cells (technical replicates merged from two independent experimental repeats): Control: 81; GSK101: 100; GSK219: 100, PEG: 100; shRNA: 102; shRNA +PEG: 104. ( K ) Like cell invasiveness, cell motility ( D ) positively scales with plasma membrane association of TRPV4 ( R ²~0.73). ( L ) Cell motility ( D ) inversely relates to cell volume ( R ²~0.89). ( M ) Cell motility ( D ) and cell invasiveness show a strong linear relationship ( R ²~0.85), enabling the use of cell motility measurements to assess overall cell invasiveness. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: p>0.05. Figure 5—source data 1. Original data corresponding to panel A. Figure 5—source data 2. Original data corresponding to panel B. Figure 5—source data 3. Original data corresponding to panel C. Figure 5—source data 4. Original data corresponding to panel D. Figure 5—source data 5. Original data corresponding to panel E. Figure 5—source data 6. Original data corresponding to panel F. Figure 5—source data 7. Original data corresponding to panel G. Figure 5—source data 8. Original data corresponding to panel I. Figure 5—source data 9. Original data corresponding to panel J. Figure 5—source data 10. Original data corresponding to panel K. Figure 5—source data 11. Original data corresponding to panel L. Figure 5—source data 12. Original data corresponding to panel M.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Inhibition, Activation Assay, Membrane, Control, Motility Assay, Diffusion-based Assay, shRNA, Single-cell Analysis

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: ( A ) In MCF10CA1a cells, plasma membrane relocation of TRPV4 was induced by a 15 min treatment with 2% PEG 300 (74.4 mOsm/kg) or by overconfluence (OC) conditions. IF images show a largely intracellular distribution of TRPV4 (red) in normal density (ND) control cells, whereas cells treated with PEG 300 or subjected to OC exhibit a significant increase in plasma membrane-associated TRPV4. Nuclei are stained with DAPI (blue). Scale bars in all panels of this figure represent 10 μm. ( B ) Line analysis quantifying plasma membrane-associated TRPV4 (%) reveals significant increases following a 1 hr treatment with GSK219 (1 nM), a 15 min exposure to 74.4 mOsm/kg PEG 300, or under OC conditions in MCF10CA1a cells. In contrast, no significant increase in plasma membrane TRPV4 is observed with GSK101 treatment. ( C ) In MCF10CA1a cells, cell movement diffusivity ( D ) increased following GSK219 or PEG 300 treatments, whereas it decreased with GSK101. Conversely, movement directionality ( v ) increased significantly with GSK101 (0.2 pM) but remained unchanged with GSK219 or PEG treatments. ( D-O ) No plasma membrane relocation of TRPV4 was observed in response to inhibition by GSK219, hyperosmotic stress induced by PEG 300, or cell crowding (OC) in MCF10AT1 ( D, E, F ), MDA-MB-231 ( G, H, I ), ETCC-006 ( J, K, L ), and ETCC-010 ( M, N, O ) cells. Similarly, GSK219 or PEG 300 did not increase single-cell motility in these cells. This is demonstrated by IF images (TRPV4: red; DAPI: blue) ( D, G, J, M ), line analysis results for plasma membrane-associated TRPV4 ( E, H, K, N ), and single-cell motility analyses for diffusivity ( D ) and directionality ( v ) ( F, I, L, O ). Notably, none of these cell lines showed motility changes in response to PEG 300 treatment. However, responses to TRPV4 activation (GSK101) and inhibition (GSK219) varied across cell types, suggesting distinct roles of TRPV4 in their cancer biology. In MCF10AT1 cells ( F ), GSK219 significantly reduced diffusivity ( D ), while no other treatment affected D or v . In MDA-MB-231 cells ( I ), neither D nor v was altered by any treatment, indicating that TRPV4 has an insignificant role in their motility. Both ETCC-006 and ETCC-010 cells exhibited increased diffusivity with GSK101; however, GSK219 also increased diffusivity in ETCC-006 cells ( L ), while having no effect on ETCC-010 cells ( O ). Directionality ( v ) increased with GSK101 in ETCC-006 cells ( L ), whereas ETCC-010 cells showed no change in v across all conditions ( O ). The number of line analyses for plasma membrane-associated TRPV4 under ND control, ND +0.2 pM GSK101, ND +1 nM GSK219, ND +2% PEG 300, and OC conditions (technical replicates merged from three independent experimental repeats) were: MCF10CA1a (B): 11, 6, 8, 12, 10; MCF10AT1 ( E ): 13, 9, 8, 9, 19; MDA-MB-231 ( H ): 13, 9, 8, 9, 19; ETCC-006 ( K ): 12, 10, 10, 5, 10; and ETCC-010 (N): 5, 5, 7, 5, 6. The number of single-cell motility analyses under ND control, ND +0.2 pM GSK101, ND +1 nM GSK219, and ND +2% PEG 300 conditions were: MCF10CA1a ( C ): 100, 100, 100, 100; MCF10AT1 ( F ): 130, 161, 582, 183; MDA-MB-231 ( I ): 57, 6, 21, 442; ETCC-006 ( L ): 65, 66, 24, 100; and ETCC-010 (O): 317, 1136, 43, 71. ( P ) Plasma membrane association of TRPV4 (% PM TRPV4) scaled positively with cell diffusivity ( D ) over a broader range in MCF10DCIS.com cells compared to MCF10CA1a cells, consistent with the higher cell volume plasticity observed in MCF10DCIS.com cells. This finding suggests that both cell types engage a pro-invasive mechanotransduction pathway. ( Q ) In contrast, this scaling relationship is absent in MCF10AT1, MDA-MB-231, ETCC-006, and ETCC-010 cells, indicating a lack of the mechanotransduction response. ( R ) The presence of this pathway in MCF10CA1a and MCF10DCIS.com cells is further supported by the observed > twofold increase in TRPV4 plasma membrane association (x-axis; PM TRPV4_peg/PM TRPV4_ctrl) and > onefold increase in diffusivity (y-axis; Dpeg/Dctrl) following PEG-300 treatment. ( S ) The cell volume reduction-driven mechanotransduction pathway is further demonstrated by plotting PEG-300-induced changes in TRPV4 plasma membrane association (x-axis; PM TRPV4_peg/PM TRPV4_ctrl) against the diffusivity ratio with GSK219 versus GSK101 (y-axis), where both cell types show a significantly greater than twofold increase, highlighting the activation of this pathway in MCF10DCIS.com and MCF10CA1a cells.****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: p>0.05. Figure 6—source data 1. Original data corresponding to panel B. Figure 6—source data 2. Original data corresponding to panel C. Figure 6—source data 3. Original data corresponding to panel E. Figure 6—source data 4. Original data corresponding to panel F. Figure 6—source data 5. Original data corresponding to panel H. Figure 6—source data 6. Original data corresponding to panel I. Figure 6—source data 7. Original data corresponding to panel K. Figure 6—source data 8. Original data corresponding to panel L. Figure 6—source data 9. Original data corresponding to panel N. Figure 6—source data 10. Original data corresponding to panel O. Figure 6—source data 11. Original data corresponding to panels P and Q. Figure 6—source data 12. Original data corresponding to panels R and S.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Membrane, Control, Staining, Inhibition, Activation Assay

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: The effects of treatment with GSK101 (0.2 pM, 1 hr), GSK219 (1 nM, 1 hr), and PEG 300 (74.4 mOsm/kg, 15-miniutes) on cells under normal density (ND) or overconfluence (OC) conditions revealed differences in TRPV4 localization (red) in the immunofluorescence (IF) images (cyan: DAPI). Only MCF10CA1a cells show GSK219, PEG 300, and OC-induced TRPV4 relocation to the plasma membrane. Other cell types, including MCF10AT1, MDA-MB-231, ETCC-06, and ETCC-10, did not exhibit this translocation. Scale bar = 20 μm.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Immunofluorescence, Membrane, Translocation Assay

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet: This cell-crowding-induced pro-invasive pathway involves a cascade of events, including ion channel inhibition, intracellular calcium reduction, cell volume reduction and cortical stiffening, and increased cell invasiveness and motility. In high-grade DCIS cells, calcium-permeable ion channels such as TRPV4 relocate to the plasma membrane upon inhibition, compensating for reduced intracellular calcium levels by priming the channels for later activation under mechanical stress. The pro-invasive mechanotransduction pathway is selectively triggered by cell crowding or hyperosmotic stress in high-grade DCIS cells, which exhibit significant TRPV4 plasma membrane relocation, pronounced cell volume reduction, and increased motility and invasiveness. In contrast, less aggressive or normal cells remain significantly less or non-responsive to these stimuli. Notably, MCF10DCIS.com cells exhibit greater cell volume reduction compared to other cells, likely due to their larger baseline cell volume at normal density, demonstrating their high cell volume plasticity that correlates with crowding-induced invasiveness. The extent of TRPV4 plasma membrane relocation, cell volume reduction, and increased invasiveness and motility scales with each other, where the increased TRPV4 association with the plasma membrane can robustly serve as a marker of pro-invasive mechanotransduction activation. This mechanotransduction capability sets high-grade DCIS cells apart from less aggressive cells, providing a critical criterion that may help identify high-risk cells with invasive potential. This mechanotransduction capability was also validated in patient specimens, suggesting its relevance in clinical settings. Future investigations will include utilizing TRPV4 localization patterns as a diagnostic tool to assist in pathological grading and as a prognostic marker to identify high-risk DCIS cells likely to activate this pathway under mechanical stress.

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Inhibition, Membrane, Activation Assay, Marker, Diagnostic Assay

Journal: eLife

Article Title: Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

doi: 10.7554/eLife.100490

Figure Lengend Snippet:

Article Snippet: Cells were seeded in a LabTek II 8-well Chambered Coverglass dish (Thermo Fisher 155409) at a density of 10,000 cells per well and allowed to adhere and grow for 2 d. Afterward, the cells were incubated on ice for 1 hr with 1:300 Anti-TRPV4 (extracellular) rabbit primary antibody (Alomone ACC-124) in either complete media or 74 mOsm/Kg PEG 300 in complete media.

Techniques: Variant Assay, Expressing, Derivative Assay, Transfection, Construct, shRNA, Plasmid Preparation, Recombinant, Affinity Purification, Bicinchoninic Acid Protein Assay, Acid Assay, Calcium Assay, Fluorescence, Infection, Immunofluorescence, Invasion Assay, Staining, Reporter Assay, Viability Assay, Mass Spectrometry, Motility Assay

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of anti-sense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerised DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). B) HCR-FISH / IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs colocalising with nuclear speckle marker SC-35 (red, SC-35 antibody), merged with DAPI staining (blue). The colocalisation of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). C) HCR-Proxy FISH micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cells nuclei were visualized with DAPI staining blue). The colocalisation of the signal was calculated using Pearson’s correlation coefficient (PCC) from 10 images (dots represent each cell). D) HCR-Proxy FISH micrographs of Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). Difference in intensity density was calculated with two-sided Student’s t-test (p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). E) HCR-Proxy FISH micrographs of Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals, merged with DAPI staining (blue).

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: In Situ, Amplification, Marker, Staining

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A) Schematic workflow of STED image acquisition platform. In each coarsely sampled confocal image of the tiled panorama scan, nuclei and RNA foci were automatically identified for subsequent super-resolution STED imaging. B) STED microscopy reveals nonspecific biotinylation. STED micrograph and intensity plot of the biotinylation signal (yellow, Alexa647-conjugated streptavidin) beyond RNA FISH boundary (red, DyLight594-conjugated anti-DIG). Reduced intensity of the biotinylation signal in the condensate’s core is barely detected with confocal microscopy. C) Schematic workflow of the fluorescence correlation spectroscopy (FCS) experiment to infer the particle diffusion dynamics (red dots present tracer molecule Alexa 647) sampled in cells’ nucleus, cytoplasm, and extracellular compartment. Autocorrelation curves shifted towards longer lag times indicate slower diffusion. Characteristic diffusion times (DT) shown in panel D were obtained by fitting experimental autocorrelation curves with a 3D diffusion model. D) Quantification of diffusion time for Alexa647 fluorophore from FCS experiment within different cellular compartments for different conditions. Average number of measurements per condition, depicted as dots: extracellular = 30, cytoplasm = 60, nucleus = 60 (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). E) Effect of molecular crowding agents on the biotinylation specificity. Top: HCR-Proxy FISH micrographs against Efl1 intronic transcript, where proximity labelling was performed with PBS, 2.5 % BSA, 5 % BSA, 40 % trehalose and Combination of BSA and trehalose (2.5 % BSA & 20 % trehalose). Bottom: Quantification of the difference in radius size between biotinylation (yellow, Alexa647-conjugated streptavidin) and RNA FISH signal (red, DyLight594-conjugated anti-DIG). Approximately 100 images per condition were acquired, depicted as dots (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). F) Effect of labelling time extension on the biotinylation specificity. Top: HCR-Proxy FISH micrographs of Efl1 transcript, where proximity labelling was performed under five different labelling times: 1, 3, 5, 15 and 30 minutes, respectively. For this experiment labelling solution Combination 1 was utilized. Bottom: Quantification of the difference (delta) in radius size between biotinylation (yellow, Alexa647-conjugated streptavidin) and RNA FISH signal (red, DyLight594-conjugated anti-DIG). Approximately 100 images per condition were acquired, depicted as dots (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *).

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Imaging, Microscopy, Confocal Microscopy, Fluorescence, Spectroscopy, Diffusion-based Assay

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A. HCR-FISH micrographs of pre-rRNA (A’ downstream), Norad and intronic Efl1 sequence (yellow, DyLight594-conjugated anti-DIG) combined with immunofluorescence detection of nucleolar marker NPM1 (red, NPM1 antibody), merged with DAPI (blue). B. UMAP analysis of nucleolar and nuclear targets from benchmarked HCR-Proxy MS experiment. C. Volcano plot for the pre-rRNA-proximal proteome. Proteins marked in red present bona fide nucleolar proteins, meanwhile literature-known nucleolar proteins are labelled (two-sided Student’s t test-based log2 fold-change > 1, fdr- adjusted p-value < 0.05). D. GO term annotation of identified bona fide nucleolar protein interactors.

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Sequencing, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A. HCR-FISH micrographs of RNA baits targeting sublayers of multiphased nucleolus (A’ upstream, A’ downstream and ITS2) (yellow, DyLight594-conjugated anti-DIG) combined with immunofluorescence detection of FC marker POLR1A, DFC marker FBL and GC marker NPM1 (red), merged with DAPI (blue). B. Radar plot showing the mean difference in amino acid usage in A′ UP, A′ DOWN, and ITS2 proximal proteomes relative to the nuclear background, with 95% confidence intervals. C. Clustermap of mean NARDINI pairwise blockiness Z-scores of eight amino acid residue classes for A′ UP, A′ DOWN and ITS2 proximal proteomes. D. Annotation analysis of RRM domain presence in A′ UP, A′ DOWN, and ITS2 proximal proteomes and nuclear control. Left: percentage of proteins containing at least one RRM domain. Right: percentage of proteins with a given number of RRM domains.

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Immunofluorescence, Marker, Residue, Control

Journal: Oncotarget

Article Title: Arg-Leu-Tyr-Glu tetrapeptide inhibits tumor progression by suppressing angiogenesis and vascular permeability via VEGF receptor-2 antagonism

doi: 10.18632/oncotarget.14343

Figure Lengend Snippet: A . Endothelial cell permeability was determined by diffusion of [ 14 C]-sucrose through HUVEC monolayers treated with 20 ng/ml of VEGF alone or in combination with RLYE (n = 3). B . Phosphorylation of VE-cadherin was determined in cell lysates of HUVECs treated with 20 ng/ml of VEGF alone or in combination with RLYE. C . Immunostaining of VE-cadherin in endothelial monolayer was determined by confocal microscopy. D-J . HCT116 tumor-bearing mice (n = 5 per group) were injected with saline or RLYE (0.5 mg/kg) for 12 days. Tumor vascular leakage was determined by the Evans blue method (E and F) or FITC-dextran image assay (G and H). (G) Immunofluorescence staining of the tumor sections (n = 5) for CD31 and VE-cadherin, and (H) ratio of VE-cadherin to CD31 was quantified using computer-aided confocal microscopy. (I) Immunofluorescence staining of the tumor sections (n = 5) for CD31 and NG2, and (J) NG2-positive vessels were quantified. K-M . HCT116 tumor-bearing mice (n = 7 per group) were i.p. injected with RLYE (0.5 mg/kg/day) alone or in combination with irinotecan (CPT-11, 17 mg/kg every 5 th day). (K) Tumor volumes were measured every 3 days. (L and M) Apoptotic cells in tumor tissues were determined and quantified by TUNEL staining. Scale bar, 100 μm in all images. ** P <0.001 (Student's t -test except survival curve data).

Article Snippet: Cells were labeled with antibodies against human VE-cadherin (Santa Cruz) and human CD31 (PECAM-1, Santa Cruz).

Techniques: Permeability, Diffusion-based Assay, Phospho-proteomics, Immunostaining, Confocal Microscopy, Injection, Saline, Immunofluorescence, Staining, TUNEL Assay

Journal: Cancer Cell International

Article Title: The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

doi: 10.1186/s12935-017-0413-y

Figure Lengend Snippet: Effect of CIGB-300 on CK2-dependent signaling pathways. a Nuclear, cytoplasmic or whole cell extracts prepared from H125 cells treated with low-lethal doses of CIGB-300 in combination or not with an activating stimulus, were resolved on 10% SDS-PAGE and blotted with p65/RelA and P-p65 (Ser529) antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. For the determination of p65 levels PMA was used as the activating stimulus, while TNFα was used for P-p65 determinations. Results are representative of three independent experiments. b Whole cell extracts prepared from H125 cells treated with CIGB-300, were resolved on 10% SDS-PAGE and blotted with CYCLIN D1, CYCLIN E and BAX antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. c H125 cells were transiently co-transfected with pNF-κB-RE-luc and pRL-TK-luc vectors, treated with low-lethal doses of CIGB-300 in combination or not with PMA and luciferase activity was determined. Data were normalized to the constitutive Renilla luciferase activity and expressed as the mean ± SD. **p < 0.01 vs. PMA treated cells and *p < 0.05 PMA + CIGB-300 treated cells vs. PMA treated cells (one-way ANOVA test). Results are representative of three independent experiments. d H125 cells were treated with low-lethal doses of CIGB-300 in combination or not with PMA. Cellular distribution of p65 was visualized by immunofluorescence microscopy. Figure shows representative images of three independent experiments, scale bar 50 μm. White arrows indicate highly positive nucleus. Representative insets of cells treated with PMA in combination or not with CIGB-300 are shown, scale bar 5 μm ( left side ). Relative p65 nuclear intensity was measured and represented as a percentage of control cells ( light gray bars ). **p < 0.01 vs. CIGB-300 treated cells (one-way ANOVA test). The percentage of those cells with highly positive nucleus ( white arrows ) was also measured ( dark gray bars ). **p < 0.01 vs. PMA treated cells and *p < 0.05 PMA + CIGB-300 treated cells vs. PMA treated cells (one-way ANOVA test) ( right side ). e H125 cells were incubated with CM containing the wnt3a factor and then treated with low-lethal doses of CIGB-300 or TBB. Cytoplasmic protein content was separated by SDS-PAGE 10% and the membrane was blotted with anti β-CATENIN antibody (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments

Article Snippet: Anti-NF-κB p65 (D14E12 XP ® , 1:1000) was purchased from Cell Signaling (Danvers, MA, USA), β-CATENIN antibody (#610,153, 1:1.000) was from BD Transduction Laboratories (San Jose, CA, USA), Horseradish peroxidase-conjugated anti-mouse (1:5000), Mouse monoclonal anti c-MYC (sc-40, 1:100) and Mouse monoclonal anti CYCLIN E (sc-247, 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Protein-Protein interactions, SDS Page, Expressing, Control, Transfection, Luciferase, Activity Assay, Immunofluorescence, Microscopy, Incubation, Membrane

Journal: Cancer Cell International

Article Title: The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

doi: 10.1186/s12935-017-0413-y

Figure Lengend Snippet: NF-κB signaling pathway status after cisplatin treatment. a H125 cells were treated with low-lethal doses CIGB-300 for 45 min in the presence or not of cisplatin. Nuclear extracts were prepared, resolved in 10% SDS-PAGE and blotted with anti p65/RelA antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments. b A549 and A549-cispR cultures were treated with cisplatin and cell number was assessed 72 h later. Viability values were compared to those obtained in both cell lines without treatment, which were set as 100%. Data expressed as the mean ± SE are representative of three independent experiments. *p < 0.05 vs. the parental cell line (Student’s t test). c Whole cell lysates prepared from A549and A549-cispR cells were resolved on 10% SDS-PAGE and blotted with anti p65/RelA antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments. d Nuclear extracts prepared from A549and A549-cispR cells treated with cisplatin were resolved on 10% SDS-PAGE and blotted with anti P65/RelA antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments

Article Snippet: Anti-NF-κB p65 (D14E12 XP ® , 1:1000) was purchased from Cell Signaling (Danvers, MA, USA), β-CATENIN antibody (#610,153, 1:1.000) was from BD Transduction Laboratories (San Jose, CA, USA), Horseradish peroxidase-conjugated anti-mouse (1:5000), Mouse monoclonal anti c-MYC (sc-40, 1:100) and Mouse monoclonal anti CYCLIN E (sc-247, 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: SDS Page, Expressing, Control

Journal: Cancer Cell International

Article Title: The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

doi: 10.1186/s12935-017-0413-y

Figure Lengend Snippet: Evaluation of Cisplatin and CIGB-300 combined treatment in a chemo-resistant setting. a A549 and A549-cispR cells were treated with low-lethal doses of CIGB-300 (150 μM) and/or Cisplatin and cell number was assessed 72 h later. Viability values were compared to those obtained in both cell lines without treatment, which were set as 100%. Data represent the mean ± SE of three independent experiments. *p < 0.05 vs. the parental cell line (one-way ANOVA test). b Chou and Talalay combination index (CI) plot for CIGB-300 and Cisplatin values of three independent experiments. c A549 and A549-cispR cells were treated with a high dose of CIGB-300 (300 μM) during 72 h and cell viability was evaluated using the MTS assay. Viability values were compared to those obtained in both cell lines without treatment, which were set as 100%. Data represent the mean ± SE of three independent experiments. *p < 0.01 vs. the parental cell line (Student’s t test). d A549 and A549-cispR cells were treated with a high dose of CIGB-300 (300 μM) during 18 h and apoptosis induction was visualized by an acridine orange/ethidium bromide double staining assay. Figure shows representative images of three independent experiments ( left side ). The number of dead cells was quantified and expressed as a percentage of the total cell number for each cell line ( right side ). Data represent the mean ± SE of three independent experiments. *p < 0.05 vs. the parental cell line (Student’s t test). e A549 and A549-cispR cells were treated with 150 μM of CIGB-300 for 90 min in the presence or not of cisplatin. PMA was used as control. Nuclear extracts were prepared, resolved in 10% SDS-PAGE and blotted with anti p65/RelA antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments. f Whole cell extracts prepared from A549 and A549-cispR cells treated with 300 μM of CIGB-300, were resolved on 10% SDS-PAGE and blotted with BAX and c-MYC antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control

Article Snippet: Anti-NF-κB p65 (D14E12 XP ® , 1:1000) was purchased from Cell Signaling (Danvers, MA, USA), β-CATENIN antibody (#610,153, 1:1.000) was from BD Transduction Laboratories (San Jose, CA, USA), Horseradish peroxidase-conjugated anti-mouse (1:5000), Mouse monoclonal anti c-MYC (sc-40, 1:100) and Mouse monoclonal anti CYCLIN E (sc-247, 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: MTS Assay, Double Staining, Control, SDS Page, Expressing

Journal: Cancer Cell International

Article Title: The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

doi: 10.1186/s12935-017-0413-y

Figure Lengend Snippet: Association between NF-κB modulation and proteasome activity. a H125 cells pretreated with a non-cytotoxic dose of Bortezomib were exposed to CIGB-300. Nuclear extracts were prepared, resolved in 10% SDS-PAGE and blotted with anti p65/RelA antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments. b Proteasome activity determination following the Proteasome-Glo™ cell-based assay for H125 treated with CIGB-300 or Bortezomib (Bz) as positive control. Data was expressed as the mean ± SE of three independent experiments. *p < 0.05 vs. Bz treated cells and ***p < 0.001 vs. control treated cells (one-way ANOVA test). c Cellular distribution of CIGB-300 ( green , FITC) and α7/C8 protein ( red , Alexa647) was visualized by confocal microscopy ( top ). Figure shows representative images of three independent experiments. Single cell analysis of Pearson and Manders coefficients were performed, as well as a representative colocalization map and a dot plot for both probes ( bottom ). Coefficient values are expressed as the mean ± SE of three independent experiments

Article Snippet: Anti-NF-κB p65 (D14E12 XP ® , 1:1000) was purchased from Cell Signaling (Danvers, MA, USA), β-CATENIN antibody (#610,153, 1:1.000) was from BD Transduction Laboratories (San Jose, CA, USA), Horseradish peroxidase-conjugated anti-mouse (1:5000), Mouse monoclonal anti c-MYC (sc-40, 1:100) and Mouse monoclonal anti CYCLIN E (sc-247, 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activity Assay, SDS Page, Expressing, Control, Cell Based Assay, Positive Control, Confocal Microscopy, Single-cell Analysis

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Top, partial amino acid sequence of the human EphA2 receptor showing the TM helix (underlined), preceded by a short extracellular segment, and followed by the start of the juxtamembrane segment. Residue numbers in the sequence of EphA2 are shown. Middle , sequence of the TYPE7 peptide, where the acidic residues introduced are shown in red. Bottom , sequence of the TMJM 563 -EphA2 peptide used in panel D. ( B ) Circular dichroism determination of TYPE7 secondary structure in buffer at pH 8 (grey line), and in the presence of POPC vesicles at pH 8 (dotted blue line) and after acidification to pH 4 (red line). ( C ) TYPE7 binding to POPC vesicles at pH 5 (red) and pH 8 (blue). Lines are fittings to , used to determine the Kp values. Lipid binding was measured using the environmentally-sensitive dye NBD attached to the N t of TYPE7. ( D ) Determination of the pH midpoint (pH 50 ) for the insertion of TYPE7 into POPC vesicles. TYPE7 data is shown in red symbols. Data obtained in vesicles containing the GWALP23 peptide control are shown in grey, and in vesicles containing TMJM 563 -EphA2 in orange. Peptide insertion was monitored by following changes in the NBD spectral center of mass ( ; ). Control OCD experiments showed that TMJM 563 -EphA2 formed a TM helix . The lines correspond to the fitting to the data using and 95% confidence intervals are shown as shaded areas ( n = 6). ( E ) OCD determination of the membrane orientation of TYPE7. Data were obtained in POPC (16:0,18:1-PC, dashed red line) and 22:1,22:1-PC (continuous red line). The theoretical spectra for a perfectly transmembrane (0°, black line) and peripheral (90°, grey line) helix are shown as a reference. ( F ) Cartoon of the different states TYPE7 (blue) adopts, and how TMJM 563 -EphA2 (orange) promotes the TM state of TYPE7. Arrows represent approximate equilibrium conditions found at pH ~6.5. The (+) symbols represent basic residues in the juxtamembrane segment of EphA2.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Sequencing, Residue, Circular Dichroism, Binding Assay, Control, Membrane

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) TYPE7-bodipy FL-X binding to H358 cells at pH 5, 6 and 7. Data at different pH values were normalized to maximum fluorescence. Mean ±S.D., n = 3. Student’s t -test; *p<0.05; **p<0.01 and NS: not significant. ( B ) H358 cells were treated with increasing concentrations of TYPE7 (0.5, 1 and 2 μM) during 24 hr. Cell viability was assessed using the MTS assay. The results indicate that TYPE7 does not cause toxicity to treated cells. Mean ±S.D., n = 3. ( C ) We threaded the sequence of TYPE7 (blue) onto one of the helices of the published dimeric structure of the transmembrane domain of EphA2 (PDB: 2K9Y) (orange). The residues substituted with glutamic acid are shown as spheres on TYPE7 outside the helix interface. The corresponding EphA2 residues are highlighted on the opposite orange helix.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Fluorescence, MTS Assay, Sequencing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) OCD spectrum of TMJM 563 -EphA2 in POPC (16:0,18:1-PC) bilayers. ( B ) HPLC data showing that TMJM 563 -EphA2 does not dimerize using a disulfide bond. Top , chromatogram showing the elution of the TMJM 563 -EphA2 monomer at 26.2 min. Bottom , control experiment where TMJM 563 -EphA2 dimerization was induced by oxidation with 10 mM copper phenanthroline for 3.5 hr. A dimeric peak appears at 20.2 min, which was not observed in the absence of oxidizing agent.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Confocal microscopy shows co-localization of TYPE7 and EphA2. A375 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 0.2 µM TYPE7-Alexa568 (red) for 5 min at room temperature. Cells were fixed and endogenous EphA2 was labeled via immunofluorescence (green). Images were collected using a 63x objective, and insets show images corresponding to the white dashed areas collected with a 100x objective. Scale bars are 20 µm and 5 µm, respectively. ( B ) The Pearson correlation coefficient (r) was calculated for cells incubated with TYPE7 in the absence and presence of EA1. Bar graph shows mean ±S.D. Student’s t -test was performed for 14 – 17 images. *p<0.05, with as effect size of 0.80 standard deviations, n = 2. ( C ) Top, SDS-PAGE showing that TYPE7-DL co-precipitates with endogenous EphA2 when using a polyclonal anti-rabbit EphA2 antibody. Middle , control Western blots of EphA2 immunoprecipitation blotted with mouse anti-EphA2 show that similar amounts of endogenous EphA2 were pulled down in all samples. Total cell lysates blotted with EphA2 and β-actin indicate that similar levels of protein were loaded. Bottom, quantification of the fluorescent bands. Bar graph shows mean ±S.D. as a percentage of maximum intensity. A Mann-Whitney test was performed (*p<0.05), n = 3.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Confocal Microscopy, Incubation, Labeling, Immunofluorescence, SDS Page, Control, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Left , cell migration was measured in the presence and absence of TYPE7 and EA1 using a Boyden cell chamber assay. Representative images are shown. Right , quantification of migrating cells, showing that incubation with TYPE7 reduced A375 cell migration to a similar degree as EA1, with effect sizes of 8.4 and 12.6 standard deviations from control, respectively. N = 3. Cells were treated with an isolated Fc group as a control for the Fc present in EA1. Scale bar is 200 µm ( B–E ), Phosphorylation of Y772 and JMS phosphorylation at Y588 and Y594. A representative Western blot is shown ( B ). Band intensity was quantified for p-Y772 ( C ), p-Y588 ( D ), and p-Y594 ( E ). We found that incubation with TYPE7 increased phosphorylation of Y772 as efficiently as EA1, with effect sizes of 5.1 and 7.7 standard deviations from control, respectively. Mean ±S.D. are shown. n = 5. ( F–H ), Phosphorylation levels of Akt. A representative Western blot is shown ( F ) and band intensity was quantified for p-T308 ( G ) and p-S473 ( H ). Lysates were blotted against total EphA2 to detect total protein levels, and β-actin as a loading control. Student’s t -test was performed to obtain p values (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 and NS, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, Boyden Chamber Assay, Incubation, Control, Isolation, Phospho-proteomics, Western Blot

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Comparison of the sequences of TYPE7 and pHLIP, with acidic residues marked in orange. Experiments were performed in A375 cells ( B ) and H358 cells ( C ). Top panels , cell lysates were blotted with anti-phospho-EphA2 Y772, and EphA2 and anti-β-actin as loading controls; Bottom panels , quantification of p-EphA2 Y772 bands. Cells were treated with Fc, TYPE7 (2 μM), pHLIP (2 μM), or EA1 (0.5 μg/mL). Statistical analysis was performed using a Student’s t -test; *p<0.05, NS = no significant differences. n = 4 – 6 for panel B, and n = 3 for panel C. All experiments were performed at pH 7.4, except pHLIP in panel B, which was performed at pH 4.2 to ensure complete TM helix formation of pHLIP.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 μg/mL), TYPE7 (2 μM) or EA1 (0.5 μg/mL). The cell lysates were blotted with anti-phospho-EphA2 S897 and anti-β-actin to assess total protein loading. ( B ) EphA2-phospho-S897 quantification of five independent experiments. Statistical analysis was performed by using a Student’s t -test, which indicated no significant differences between samples and controls. ( C ) MTS cell proliferation assay. A375 cells were treated with Fc (0.5 μg/mL), EA1 (3 μg/mL), TYPE7 (2 μM) and TYPE7 +EA1 for 48 hr. No significant differences between Fc control and TYPE7 treated cells were found using a Student’s t -test; **p<0.01. Mean ±S.D., n = 3. ( D–E ) EphA2 expression levels do not change after TYPE7 treatment. Student’s t -test was performed and no significant differences were found between samples. Mean ±S.D., n = 5.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Proliferation Assay, Control, Expressing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 mg/mL), TYPE7 (2 μM), pHLIP (2 μM) or EA1 (0.5 mg/mL). After treatment, cell lysates were incubated overnight with array membranes to detect tyrosine phosphorylation of 49 different RTKs. The three pairs of reference spots used for blot alignment are boxed pink. Boxed RTK are: EphA1 (blue), EphA2 (yellow), HGFR/c-MET (green), EGFR (red) and ErbB3 (orange). ( B ) Bar graph shows mean and standard deviation of selected RTKs. The table on the right shows the identity of all the RTKs. ( C ) pHLIP weakly promotes phosphorylation of ErbB3 and HGFR/c-MET, as TYPE7 does. Since pHLIP does not induce EphA2 phosphorylation at Y772 or affects cell migration , this evidence logically argues against activation of those RTKs being involved in the TYPE7 regulation of these events.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Phospho-proteomics, Standard Deviation, Migration, Activation Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Super-resolution SIM data. H358 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 2 µM TYPE7. Representative images show fluorescence obtained using an anti-EphA2 antibody ( n = 4). Scale bar is 10 µm. Insets magnify areas with clusters, and the scale bars are 5 µm. ( B ) Representative FCS autocorrelation curves for EphA2FL-GFP in control conditions (green) or in the presence of TYPE7 (blue) and EA1 (magenta). Δτ1 and Δτ2 represent the changes in dwell time. ( C–E ) Diffusion coefficient results, containing graphic models describing the EphA2 constructs used. ( C ) Box-whisker plot of measurement of the FCS diffusion coefficient of EphA2FL-GFP. ( D ) Diffusion coefficient of EphA2ΔJ-GFP. ( E ) Diffusion coefficient of Myr-EphA2 ICD-GFP. Diffusion coefficients collected from cells with and without TYPE7 treatment are reported along with EA1 ligand stimulation (orange boxes). The median values are reported next to the box plots. Each data point is the average of five 10 s FCS measurements on one cell. The grey numbers on top of the plots are the total number of cells measured. Criteria for the box, median, quartiles, whiskers and outliers are described elsewhere . One-way ANOVA tests were performed to obtain the p values (****p<0.0001; ns, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Fluorescence, Control, Diffusion-based Assay, Construct, Whisker Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) FCS experiments. Schematic diagram of a FCS experiment. A 488 nm laser beam is focused at the peripheral membrane area of a cultured cell to excite the GFP tag on the diffusive receptors. The emitted photons are collected through the objective and directed to an avalanche photodiode (APD). The fluorescence fluctuation caused by the diffusion of receptors is recorded and transformed into the auto-correlation function. Insert: epi-fluorescence image of DU145 cell expressing GFP-tagged receptors; the red dot represents the position of laser beam. Scale bar is 5 μm. In the auto-correlation curve, τ D and G(0) report on the mobility and the concentration of the diffusive receptors, respectively. ( B ) FCS auto-correlation curves for the three EphA2 constructs. Three curves are shown for each experimental condition. ( C ) Receptor density of EphA2FL-GFP in DU145 cell membranes. Median density value is reported for EphA2FL-GFP and EphA2ΔJ-GFP. Each data point is the average of five 10 s FCS measurements on one cell. 52 cells were measured. ( D ) Representative epi-fluorescence images of cells used for FCS measurements under different conditions of TYPE7 and EA1 treatment. Scale bars are 5 μm.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Membrane, Cell Culture, Fluorescence, Diffusion-based Assay, Transformation Assay, Expressing, Concentration Assay, Construct

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of AQP8 knockdown on hydrogen peroxide diffusion through the plasma membrane (NES), the outer mitochondrial membrane (IMS), and the inner mitochondrial membrane (MLS) of HeLa cells. ( A , D , G ) The left and right panels display representative frames extracted from videos illustrating the time course of H 2 O 2 diffusion into mock-transfected (Ctr) and AQP8-knockdown (siRNA AQP8) HeLa cells before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is shown in pseudocolor in the upper panel, with the scale indicated in the insert. ( B , E , H ) The time course of H 2 O 2 fluorescence in mock- and siRNA-transfected HeLa cells is presented, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , F , I ) Computerized least squares regression analysis was employed to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the H 2 O 2 time course curves were fitted using a one-phase exponential association equation. a, p < 0.05 compared to Ctr (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Clinical Proteomics, Membrane, Transfection, Fluorescence

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of double knockdown of AQP3/AQP6/AQP8 and S1R in the hydrogen peroxide diffusion through the plasma membrane (NES) of HeLa cells. ( A ) The left and right panels display representative frames extracted from videos illustrating the time course of H 2 O 2 diffusion into HeLa cells that were single knockdown for AQP (siRNA AQP3/AQP6/AQP8) and HeLa cells that were double knockdown for AQP and S1R (siRNA AQP3/AQP6/AQP8 +S1R) before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is shown in pseudocolor in the upper panel, with the scale indicated in the insert. ( B , D , F ) The time course of H 2 O 2 fluorescence in HeLa cells with a knockdown of AQP and in cells with a double knockdown of AQP and S1R is presented, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , E , G ) Computerized least squares regression analysis was performed to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points from the time courses of H 2 O 2 curves were fitted using a one-phase exponential association equation. a, p < 0.05 compared to control (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Clinical Proteomics, Membrane, Fluorescence, Control

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Effect of double knockdown of AQP6/AQP8 and S1R on hydrogen peroxide diffusion through the inner mitochondrial membranes (MLS) of HeLa cells. ( A ) The left and right panels show representative frames extracted from videos displaying the time course of H 2 O 2 diffusion into HeLa cells that were single knockdown for AQP (siRNA AQP6/AQP8) and HeLa cells that were double knockdown for AQP and S1R (siRNA AQP6/AQP8 + S1R) before and after the addition of 50 μM H 2 O 2 , respectively. The increase in HyPer7 fluorescence is depicted in pseudocolor in the upper panel, with the scale shown in the insert. ( B , D ) The time course of H 2 O 2 fluorescence in HeLa cells knocked down for AQP and in those knocked down for both AQP and S1R is shown, starting from the addition of 50 μM H 2 O 2 . Data represent the mean of at least three different experiments, with standard deviations omitted for clarity. ( C , E ) Computerized least squares regression analysis was used to determine the k relative initial rate values (GraphPad Prism 4.00, 2003). The experimental points of the time courses of H 2 O 2 curves were fitted with a one-phase exponential association equation. a, p < 0.05 compared to control (Student’s t -test).

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Knockdown, Diffusion-based Assay, Fluorescence, Control

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: Representative immunofluorescence confocal microscopy images of colocalization of AQP8 and AQP11 with TOMM20 in HeLa cells. ( A , C ) Green labeling indicates the presence of TOMM20, and red labeling indicates the expression of AQP8 or AQP11 (Hoechst (blue) counterstained nuclei). Yellow labeling shows colocalization signal of AQP8 or AQP11 with TOMM20. Scale bar, 5 μm. ( B , D ) The graph, measured in the white line position in panels ( A , C ), shows the fluorescence signals of AQP8 or AQP11 and TOMM20 staining.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Expressing, Fluorescence, Staining

Journal: Cells

Article Title: Sigma1 Receptor Modulates Plasma Membrane and Mitochondrial Peroxiporins

doi: 10.3390/cells14141082

Figure Lengend Snippet: A possible schematic model of the functional AQPs in the plasma membrane and in the outer and inner mitochondrial membranes. AQP3, AQP6, and AQP8 are functionally active in the plasma membrane, AQP6 is functionally active in the outer mitochondrial membrane, while AQP6 and AQP8 are functionally active in the inner mitochondrial membrane. Arrows indicate the modulation of AQP8 by S1R, while the question mark indicates the uncertain modulation of AQP6.

Article Snippet: The antibodies used were the following: affinity pure anti-AQP3 rabbit antibody (ab125045, 1:200 dilution; Abcam, UK), anti-AQP6 rabbit polyclonal IgG (AQP61-A, 1:500 dilution; Alpha Diagnostic International, San Antonio, TX, USA), anti-AQP8 rabbit antibody (PA1511, 1:200 dilution; BOSTER, Pleasanton, CA, USA), anti-AQP11 rabbit antibody (PB10044, 1:100 dilution; BOSTER, Pleasanton, CA, USA), and anti-TOMM20 [4F3] (ab56783, 1:500 dilution; Abcam, UK).

Techniques: Functional Assay, Clinical Proteomics, Membrane

Journal: Science Advances

Article Title: The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia

doi: 10.1126/sciadv.abl8952

Figure Lengend Snippet: ( A ) Representative Western blotting showing CCRL2 knockdown effect by two different shRNAs (sh1 and sh2) on JAK2 (Tyr 1007/1008 ), STAT3 (Tyr 105 ), and STAT5 (Tyr 694 ) phosphorylation in MDS92 and MDS-L. ( B ) CCRL2 knockdown decreases the RNA levels of the JAK2/STAT target genes: MYC (MDS92: P = 0.001-sh1, P = 0.002-sh2; MDS-L: P = 0.001-sh1, P = 0.010-sh2), PIM1 (MDS92: P = 0.003-sh1, P = 0.010-sh2; MDS-L: P = 0.002-sh1, P = 0.040-sh2), BCL2 (MDS92: P = 0.020-sh1, P = 0.060-sh2; MDS-L: P = 0.008-sh1, P = 0.005-sh2), MCL1 (MDS92: P = 0.025-sh1, P = 0.004-sh2; MDS-L: P = 0.004-sh1, P = 0.019-sh2), and DNMT1 (MDS92: P = 0.009-sh1, P = 0.010-sh2; MDS-L: P = 0.008-sh1, P = 0.040-sh2), n = 3. ( C ) Western blotting showing that CCRL2 knockdown suppresses the phosphorylation of JAK2, STAT3, and STAT5 at 30 min and 6 hours of IL-3 (20 ng/ml) treatment following 48 hours of IL-3 starvation. ( D ) Coimmunoprecipitation assay showing that CCRL2 precipitates with JAK2 and that CCRL2 knockdown does not affect the interaction between JAK2 and the common β signal transducing subunit of CD123 (CSF2RB) but decreases the interaction between JAK2 and STAT3/5 proteins. IgG, immunoglobulin G. ( E ) Representative images from immunofluorescence staining (×40 and ×60 magnification) showing localization of CCRL2 (green) in the cytoplasm and membrane of MDS-L cells. Confocal microscopy reveals areas of colocalization with JAK2 (red). DAPI, 4′,6-diamidino-2-phenylindole. ( F ) The mean fluorescence intensity (MFI) of phosphorylated STAT3 (P-STAT3) is positively associated with the MFI of CCRL2 in CD34 + cells from patients with MDS and CD34 + blasts from patients with AML (Coef, 0.15; P = 0.001), n = 16.

Article Snippet: Cell lysates were incubated overnight with Sepharose bead conjugate JAK2 monoclonal antibody (Cell Signaling Technology, #4089) or Sepharose bead conjugate isotype control (Cell Signaling Technology, #3423).

Techniques: Western Blot, Knockdown, Phospho-proteomics, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Membrane, Confocal Microscopy, Fluorescence

Journal: Science Advances

Article Title: The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia

doi: 10.1126/sciadv.abl8952

Figure Lengend Snippet: ( A ) CCRL2 knockdown with two different lentiviruses significantly suppresses the growth of TF-1 cells in the presence ( P < 0.001) and absence of GM-CSF ( P < 0.001). CCRL2 knockdown with two different lentiviruses suppresses at a lower extent the growth of DAMI cells ( P = 0.009-sh1, P = 0.058-sh2). ( B ) CCRL2 knockdown with two different lentiviruses decreases the colony formation of TF-1 cells in the presence ( P < 0.001) and absence of GM-CSF ( P < 0.001). CCRL2 knockdown with two different lentiviruses decreases the colony formation of DAMI cells ( P = 0.002-sh1, P = 0.015-sh2) at a lower extent compared with TF-1 cells. ( C ) CCRL2 knockdown with sh1 increased the percentage of apoptotic TF-1 cells ( P = 0.015-sh1, P = 0.097-sh2), and CCRL2 knockdown with the two lentiviruses decreased the percentage of TF-1 cells in the G 2 -S phase ( P < 0.001). ( D ) CCRL2 knockdown with two different lentiviruses increases the expression of CD41 ( P < 0.001), CD71 ( P < 0.001), and CD235a ( P = 0.002-sh1, P = 0.025-sh2). CCRL2 knockdown does not affect the expression of CD41 ( P = 0.230-sh1, P = 0.912-sh2). CCRL2 knockdown increases the expression of CD71 ( P < 0.001-sh1, P = 0.026-sh2) and CD235a ( P = 0.002-sh1, P = 0.067-sh2). ( E ) Western blotting showing the effect of CCRL2 knockdown in the JAK2/STAT signaling in TF-1 and DAMI cells. CCRL2 knockdown suppresses the phosphorylation of JAK2, STAT3, and STAT5 in TF-1 cells. CCRL2 knockdown decreases the phosphorylation of JAK2 but does not affect the phosphorylation of STAT3 and STAT5 in DAMI cells.

Article Snippet: Cell lysates were incubated overnight with Sepharose bead conjugate JAK2 monoclonal antibody (Cell Signaling Technology, #4089) or Sepharose bead conjugate isotype control (Cell Signaling Technology, #3423).

Techniques: Knockdown, Expressing, Western Blot, Phospho-proteomics

Journal: Cell metabolism

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

doi: 10.1016/j.cmet.2018.09.020

Figure Lengend Snippet: (A) Gene expression levels of glucose transporters (Glut1 and Glut3) and the key enzymes in glycolysis (HK2, GPI, PFK1, TPI, ENO1, PKM2 and LDHα) in different T cell subsets. Th1, Th2 and Th17 cells were polarized from naïve T cells purified from healthy donors in the presence of related polarization cytokine conditions. nTreg cells were directly purified from PBMCs of healthy donors. Total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression level and adjusted to the levels in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent donors. (B) Gene expression levels of key enzymes in cholesterol synthesis (HMGCR, HMGCS1, SQLE, and IDI1), as well as fatty acid oxidation (CPT-1) and synthesis (ACC1 and FASN) in different T cell subsets. Cell preparations and assays were identical to (A). (C) and (D) Tumor-derived CD4+ Treg and γδ Treg cells had higher gene expression levels of glucose transporters and the key enzymes in glycolysis (in C) and lipid metabolism (in D) than those of naïve CD4+ and Th1 cells. Tumor-derived Treg cells: CD4 TregE1 is a melanoma-specific Treg cells and γδ Treg31 & 76 cells are derived from TILs of breast cancer patients. Relative mRNA expression level of each gene was determined by real-time PCR, normalized to β-actin expression and then adjusted to the level in naïve CD4+ T cells. (E) nTreg cells produced higher amounts of the key metabolites involved in the glycolysis and tricarboxylic acid cycle than other T cell subsets. Th1, Th2, Th17 and nTreg cells were prepared as (A). The cell lysates from different T cell subsets were extracted and analyzed using a LCtriple quadruple mass spectrometry for determination of cellular glucose metabolites. Metabolite levels are normalized to naïve CD4 cell group. Relative levels of intermediate metabolites in the glycolysis and TCA-cycle pathways are shown as mean ± SD from representative of three independent T cell subsets with similar results. (F) and (G) Inhibition of glycolysis and lipid metabolism dramatically blocked Treg cell suppressive capacity on T cell proliferation (in F) and prevented Treg-induced responder T cell senescence (in G). nTreg cells were pretreated with pharmacological glucose transporter, glycolysis and lipid metabolism inhibitors for 48 hours, including phloretin (2 μM), 2-DG (1 mM), LND (125 μM), and 3BrPA (30 μM), etomoxir (100 μM), C75 (5 μM), orlistat (10 μM), 25-HC (0.25 μg/ml), simvastatin (2 μM), respectively. Naïve CD4+ T cells were then co-cultured with inhibitor-pretreated or untreated Treg cells for 3 days. Proliferation of co-cultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays, and SA-β-Gal expression in treated T cells was also determined. Data shown are mean ± SD from representative of three independent experiments with similar results.* p<0.05 and ** p<0.01, compared with the medium only group.

Article Snippet: Glut1 (H-43) antibody , Santa Cruz Biotechnology , Cat# sc-7903; RRID:AB_2190936.

Techniques: Gene Expression, Purification, Isolation, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Produced, Mass Spectrometry, Inhibition, Cell Culture

Journal: Cell metabolism

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

doi: 10.1016/j.cmet.2018.09.020

Figure Lengend Snippet: (A) and (B) Poly-G3 treatment down-regulated gene (in A) and protein (in B) expressions of Glut1 and Glut3 in human Treg cells. Treg and control CD4+CD25− effector cells were treated with Poly-G3 (3 μg/ml) for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in untreated T cells (in A). Treated nTreg cells were also determined for Glut1 and Glut3 protein expression using the flow cytometry analysis (in B). Data shown in histograms are representative of average of three independent experiments ± SD. *p<0.05 and **p<0.01, compared with the medium only group. (C) Decreased Glut1 and Glut3 protein expression was induced by Poly-G3 treatment in nTreg cells but not in control CD4+ T cells after 3-day culture. Glut1 and Glut3 (green) expression was determined by an indirect immunofluorescence assay with a confocal microscopy. Scale bar, 50 μm. Results shown in the right histograms are mean ± SD of fluorescence intensity (MFI) quantifications of glucose transporters from three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (D) Poly-G3 treatment down-regulated Glut1 and Glut3 membrane expression and promoted its intracellular translocation in nTreg cells. Cell treatment and procedure were identical to (C). Percentages of glucose transporter expression in cell membrane or intracellular were counted and shown in the right histograms. Scale bar, 25 μm. Results are mean ± SD of positive cells from three independent experiments. **p<0.01, compared with the medium only group. (E) and (F) Inhibition of glucose transport significantly promoted the Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in E) and induction of cell senescence (in F). nTreg cells were pretreated with or without glucose transporter inhibitor phloretin (2 μM) for 2 days, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation of co-cultured naïve T cells stimulated with anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in E), and SA-β-Gal expression in treated T cells was determined (in F). Data shown are mean ± SD from three independent experiments with similar results. **p<0.01 between the comparison groups.

Article Snippet: Glut1 (H-43) antibody , Santa Cruz Biotechnology , Cat# sc-7903; RRID:AB_2190936.

Techniques: Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Fluorescence, Membrane, Translocation Assay, Inhibition, Cell Culture, Comparison

Journal: Cell metabolism

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

doi: 10.1016/j.cmet.2018.09.020

Figure Lengend Snippet: (A) Increased senescent cell populations were markedly induced in pre-activated CD8+ T cells after cotransfer with nTreg cells but not with control effector CD4+ T cells. Naïve CD8+ T cells (5 × 106/mouse), expanded nTreg (3 × 106/mouse) and CD4+CD25− T cells (3 × 106/mouse) were pre-activated with antiCD3 antibody and adoptively co-transferred into NSG mice. Blood and Spleens were harvested at 12 days post-injection. The transferred human CD8+ T cells were isolated for subsequent SA-β-Gal staining. **p<0.01, compared with the groups co-transferred with CD4+CD25− T cells or CD8+ T cells alone. (B) Treatment with Poly-G3, 2-DG, or 2-ME significantly prevented induction of senescence in transferred CD8+ T cells. nTreg cells were pretreated with Poly-G3 (3 μg/mL), glycolytic metabolism inhibitor 2-DG (1 mM) or HIF1α inhibitor 2-ME (10 μM) for 24 hours prior to adoptively transfer into the mice. After adoptive transfer of T cells into the mice, Poly-G3 (50 μg/mouse), 2-DG (10 mg /mouse), or 2-ME (0.3 mg /mouse) were intraperitoneally injected into mice for a total of 3 doses with 3-day intervals. Cell preparation and injection procedures were the same as in (A). The transferred human CD8+ T cells in different organs were isolated at 12 days post-injection for subsequent SA-β-Gal staining. **p<0.01, compared with the medium group. (C) and (D) Real-time PCR quantification of expression changes of glucose transporters and glycolytic enzymes in purified nTreg cells from blood and spleens of NSG mice treated with Ploy-G3 and different inhibitors. Cell treatment and adoptive transfer procedure were identical to (B). The transferred nTreg cells were isolated for RT-PCR analyses.*p<0.05 and **p<0.01, compared with the co-transferred group without inhibitor treatment. (E) to (G) Treatments with Poly-G3 and inhibitors prevented tumor-specific T cell senescence and enhanced antitumor immunity in NSG mice. Human 586mel tumor cells (5 × 106/mouse) were subcutaneously injected into NSG mice. Tumorspecific CD8+ TIL586 cells (5 × 106/mouse) were i.v. injected on day 5 with or without nTreg cells (3 × 106/mouse). In addition, nTreg cells were pretreated with TLR8 ligand Poly-G3, glycolytic metabolism inhibitor 2-DG and HIF1α inhibitor 2-ME for 24 hours prior to adoptively transfer into the mice, and mice were then intraperitoneally injected with Poly-G3, 2-DG or 2-ME for a total of 3 doses with 3-day intervals after T cell transfer. The treatment procedures and doses were identical to the experiments in (B). Tumor volumes were measured and presented as mean ± SD (in E) (n=5 mice per group). Blood, spleens, and tumors were harvested at day 33 post injection. The transferred human TIL586 T cells in different organs were isolated for SA-β-Gal staining. *p<0.05 and **p<0.01, compared between the comparison groups (in E), or compared with the groups of TIL586 alone (in F) or co-transferred group without inhibitor treatment (in G), respectively. (H) to (J) Real-time PCR quantification of glucose transporter and glycolytic enzyme expression changes in purified Treg cells from TILs, blood and spleens of tumor-bearing NSG mice treated with Ploy-G3 and different inhibitors. Cell treatment and adoptive transfer procedure were identical to (E). The transferred nTreg cells were isolated from different organs and tumor tissues for RT-PCR analyses.*p<0.05 and **p<0.01, compared with the co-transferred group without inhibitor treatment.

Article Snippet: Glut1 (H-43) antibody , Santa Cruz Biotechnology , Cat# sc-7903; RRID:AB_2190936.

Techniques: Control, Injection, Isolation, Staining, Adoptive Transfer Assay, Real-time Polymerase Chain Reaction, Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Cell metabolism

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

doi: 10.1016/j.cmet.2018.09.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Glut1 (H-43) antibody , Santa Cruz Biotechnology , Cat# sc-7903; RRID:AB_2190936.

Techniques: Derivative Assay, Recombinant, Reverse Transcription, Selection, Cell Based Assay, Software

Journal: Mucosal immunology

Article Title: Broadly neutralizing antibodies suppress post-transcytosis HIV-1 infectivity.

doi: 10.1038/mi.2016.106

Figure Lengend Snippet: Figure 1 IgA class switching of HIV-1 bNAbs. (a) Cytograms show the binding of 3BNC117 IgG1 (green), IgA1 (blue), and IgA2 (red) antibodies (as examples) to uninfected MT4C5 T cells (upper part), and to Gag þ (PE) MT4C5 cells infected with the NLAD8 viral strain (bottom part). (b) Bar graph shows the binding of the selected bNAbs expressed as IgG1 (green), IgA1 (blue), and IgA2 (red) to Gag þ infected MT4C5 cells. Horizontal bars represent the relative mean fluorescence intensity (rMFI). Error bars indicate the s.e.m. of triplicate values from two independent experiments. (c) ELISA graphs comparing the binding of YU-2 gp140 to IgG1 (green), IgA1 (blue), and IgA2 (red) bNAbs. The x-axis shows the gp140 concentration (nM) required to obtain the ELISA values (OD405 nm) indicated on the y-axis. Error bars indicate the s.e.m. of duplicate values. (d) SPR sensorgrams showing the binding of bNAbs expressed as IgG1 (green), IgA1 (blue), and IgA2 (red) to YU-2 gp140 or gp120 ligands expressed as normalized response units (RU) over time. (e) Heat map (expressed as % binding to wild-type gp120) summarizing the binding of IgG1, IgA1, and IgA2 bNAbs to selected mutant protein antigens (see Supplementary Figure 2 online). Darker colors ¼ stronger inhibition; light colors ¼ moderate inhibition. (f) ELISA graph shows binding of 10E8 IgA1 and IgA2 antibodies to MPER peptide. The x-axis shows the antibody concentration (nM) required to obtain the ELISA values (OD405 nm) indicated on the y- axis. Error bars indicate the s.e.m. of duplicate values. bNAbs, broadly neutralizing antibodies; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; MPER, membrane proximal external region; SPR, surface plasmon resonance.

Article Snippet: Secondary antibody labeled with Cy3-conjugated goat anti-mouse IgG1 (1:800 final dilution, Jackson Immunoresearch).

Techniques: Binding Assay, Infection, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Mutagenesis, Inhibition, Membrane, SPR Assay

Journal: Mucosal immunology

Article Title: Broadly neutralizing antibodies suppress post-transcytosis HIV-1 infectivity.

doi: 10.1038/mi.2016.106

Figure Lengend Snippet: Figure 3 In vitro transcytosis of HIV-1 and bNAbs. (a) Graph shows the transepithelial electrical resistance (TER) values of HEC-1A epithelial cell monolayers according to time of culture. Mean values±s.e.m. of two representative experiments (in duplicate) are shown. Transcytosis experiments were performed at day 6 (virus and black arrow). Dotted line indicates the threshold (400 Ohms cm 2) to obtain monolayer confluence and integrity of cells. (b) Immunofluorescence staining of HEC-1A monolayers with DAPI and anti-human E-cadherin antibody at day 6 and 9 of culture. Representative images are shown. Scale bar indicates 10 mm. (c) Bar graph comparing the diffusion of FITC-labeled DEAE-dextran molecules to the basal compartment when passing through the transwell membrane (CTR, control) and the HEC-1A cell monolayer at day 6. Mean values±s.e.m. of two representative experiments (in duplicate) are shown. (d) Bar graph shows the percentage of cell-free virus detected in the basal compartment compared to the input placed in the apical part of the transwell in presence of HEC-1A cells monolayer (transcytosed) or not (filtered). The gray bar corresponds to values for NLAD8 transcytosis (as a percentage of transcytosed viruses) normalized by the filtered virus amount. Mean values±s.e.m. of four representative experiments (in triplicate) are shown. (e) Bar graph shows the normalized transcytosis of NL-DEnv alone, and in presence of anti-gp120 10-1074 IgG1 (green) and non-HIV-1 reactive mGO53 control IgG1 (gray) antibodies. Mean values of duplicate obtained in three independent experiments are shown. Error bars indicate the s.e.m. Groups were compared to mGO53 negative control using Mann-Whitney test. ns, not significant. (f) Graphs show the ELISA detection post-transcytosis of IgG (green) and IgA (blue) antibodies in the apical and basal compartments. Error bars indicate the s.d. of duplicate measurements. Table in the right-hand side indicates the calculated percent of antibody transcytosis for mGO53 and NIH45-46 IgG and IgA antibodies. (g) Representative images corresponding to the 3D-reconstruction of the confocal microscopy experiments made on HEC-1A cell monolayers with GFP- labeled HIV-1 (green) in presence of mGO53, NIH45-46 or 3BNC117 (red). Nuclei and intercellular junctions were stained with DAPI (blue) and anti-E- cadherin antibody (gray), respectively. (h) Dot plot shows the number of fluorescent objects corresponding to HIV-1 alone (NL4.3), 3BNC117 IgG alone and virus-antibody complexes (Coloc) according to the distance across the Z-stack depth. Quantification was made using one representative 6 mm Z-stack acquisition. (i) Graphs show the percentage of overlap between fluorescent HIV-1 and antibodies (left-hand side), and calculated relative frequency according to bin center (right-hand side). Quantification was made using 9-12 different 2 mm Z-stack acquisitions per condition (field). (j) Dot plot shows the percentage of colocalization between virus and antibodies calculated from (i). ****P value o0.0001 (Mann-Whitney test). bNAbs, broadly neutralizing antibodies; DAPI, 4,6-diamidino-2-phenylindole; DEAE, diethylaminoethyl; Ig, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein.

Article Snippet: Secondary antibody labeled with Cy3-conjugated goat anti-mouse IgG1 (1:800 final dilution, Jackson Immunoresearch).

Techniques: In Vitro, Virus, Immunofluorescence, Staining, Diffusion-based Assay, Labeling, Membrane, Control, Negative Control, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: Mucosal immunology

Article Title: Broadly neutralizing antibodies suppress post-transcytosis HIV-1 infectivity.

doi: 10.1038/mi.2016.106

Figure Lengend Snippet: Figure 4 HIV-1 transcytosis in presence of bNAbs. (a) Bar graph shows the percent of transcytosis of NLAD8 alone (No Ab, white), in presence of non- HIV-1 mGO53 control (gray) and selected bNAbs expressed as monomeric IgG1 (green), IgA1 (blue), and IgA2 (red) antibodies. Dotted line indicates an arbitrary threshold corresponding to a twofold decrease of the values obtained with mGO53 control antibody. Mean values±s.e.m. of three to five independentexperimentsperformedinduplicateareshown.(b)Bargraphshowsthepercentoftranscytosisasin(a)butwithincreasingamountof10-1074 IgG antibodies. Mean values±s.e.m. of three independent experiments performed in duplicate are shown. (c) Bar graph shows the percent of transcytosis as in (a) for 10-1074 IgA1 but with decreasing amount of NLAD8 virus used as input (as indicated above bars). Mean values of duplicate are shown. Error bars indicate the s.e.m. (d) Bar graph shows the percent of transcytosis as in (a) but with virus in presence of monomeric (Mo), dimeric (Di) and multimeric (Mu) 10-1074 IgA1 antibodies. Mean of triplicate values are shown. Error bars indicate the s.e.m. On the left-hand side, FPLC chromatogram shows the protein separation of IgAmonomers, dimers,andmultimers by size exclusion chromatography. Thex-axisshows theelution volume(eV)required to obtain the values of absorption units at 280 nm (mAU) indicated on the y-axis. Light blue bars indicate selected fractions. (e) Bar graph shows the percent of transcytosis of as in (a) but using YU-2 viral strain. Mean values±s.e.m. of four independent experiments performed in duplicate are shown. (f) Bar graphs show the percent of transcytosis as in (a) but using cell-free and cell-produced NL4.3 viruses. Mean values±s.e.m. of two independent experiments performed in duplicate are shown. (g) Graph shows the TER values of T84 and Caco-2 epithelial cell monolayers according to time of culture. Mean values±s.e.m. of three independent experiments (more than50 transwells in total) are shown. Transcytosis experiments were performed at days 8 and 10 forCaco-2 andT84cells,respectively (virusandblackarrows).Dotted lineindicated thethreshold(1,000 Ohms cm 2) to obtainmonolayerconfluenceand integrity of cells. (h) Immunofluorescence staining of T84 and Caco-2 monolayers with DAPI and anti-human E-cadherin antibody at days 8 and 10 of culture. Representative images are shown. Scale bar indicates 20 mm. (i) Bar graph shows the percent of transcytosis of as in (a) but using T84 and Caco-2 monolayers and IgG bNAbs only. Mean values±s.e.m. of two independent experiments performed in triplicate are shown. Groups were compared to mGO53 negative control using Mann-Whitney test for all except (b), and using one-way ANOVA test for (b). ANOVA, analysis of variance; bNAbs, broadly neutralizing antibodies; FPLC, fast protein liquide chromatography; Ig, immunoglobulin; ns, not significant; TER, transepithelial electrical resistance.

Article Snippet: Secondary antibody labeled with Cy3-conjugated goat anti-mouse IgG1 (1:800 final dilution, Jackson Immunoresearch).

Techniques: Control, Virus, Size-exclusion Chromatography, Produced, Immunofluorescence, Staining, Negative Control, MANN-WHITNEY, Chromatography

Journal: Mucosal immunology

Article Title: Broadly neutralizing antibodies suppress post-transcytosis HIV-1 infectivity.

doi: 10.1038/mi.2016.106

Figure Lengend Snippet: Figure 5 Infectivity of transcytosed HIV-1. (a) Scatter plot shows the infectivity of transcytosed NLAD8 alone (No Ab, white), in presence of non-HIV-1 mGO53 control (light gray), 2F5 IgG1 bNAb (dark gray) and selected bNAbs expressed as monomeric IgG1 (green), IgA1 (blue), and IgA2 (red) antibodies on TZM-bl reporter cells. Antibodies were tested at a final concentration of 66.7 nM (originating from experiments in Figures 4a). (b) Same as in (a) but with IgG1 bNAbs only in contact with YU-2 viruses used as viral input for transcytosis. (c) Same as in (a) but with increasing concentration of 2F5 IgG antibodies (from 2.5 to 80 mg ml 1) in contact with NLAD8 virions. (d) Upper graph shows the percent of transcytosis of 3BNC117 and 10-1074 IgG1 (green), IgA1 (blue), and IgA2 (red) bNAbs tested across a large concentration range (from 0.023 to 66.7 nM). Mean values±s.e.m. of duplicate data are shown. Lower graph shows the percent of post-transcytosis neutralization of 3BNC117 and 10-1074 IgG1, IgA1, and IgA2 antibodies as tested above. (e) Table indicating IC50 values obtained after viral transcytosis (left), and calculated ratios (right) comparing them to IC50 values determined using the traditional neutralization assay. (f) Same as in (a) but with IgG1 bNAbs only in transcytosis assays using T84 and Caco-2 epithelial cell monolayers. Data represent mean of triplicate values obtained for each transcytosis point performed as shown in Figure 4. Error bars indicate the s.e.m. Groups were compared to mGO53 negative control using Mann-Whitney test for all except (c), and using one-way ANOVA test for (c). ***Po0.001; ****Po0.0001. ANOVA, analysis of variance; bNAbs, broadly neutralizing antibodies; Ig, immunoglobulin; ns, not significant.

Article Snippet: Secondary antibody labeled with Cy3-conjugated goat anti-mouse IgG1 (1:800 final dilution, Jackson Immunoresearch).

Techniques: Infection, Control, Concentration Assay, Neutralization, Negative Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: Expression levels were quantified for a colorectal cancer cell line panel using Affymetrix U133+2 mRNA microarray data. Measurements indicated three candidate cell lines, SW620, COLO320HSR and COLO741 (labelled in red, top of panel), as having very low levels of native EGFR expression, as tested in subsequent western blot analysis in comparison to EGFR-expressing cell lines as positive controls (indicated as red columns, middle and bottom of panel). Three candidate cell lines with very low or absent levels of EGFR mRNA (SW620, COLO320HSR, COLO741; Y axis text label in red, top of panel) and a further four positive controls with medium to high levels (HCT116, LS180, COLO678; indicated as red columns, middle and bottom of panel), were selected and protein levels confirmed by western blot .

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Expressing, Microarray, Western Blot, Comparison

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Dual-colour TIRF applied to EGFR-GFP transfected human colorectal carcinoma cells with and without presence of fluorescently-labelled EGF-TMR. Several models to explain EGF activation of EGFR have been postulated, including ‘monomer’ and ‘preformed dimer’ models (EGFR structure PDB ID 1egf; EGFR monomer and dimer cartoons have been generated by manually combining separate structures with PDB ID values of 1nql, 1ivo, 2jwa, 1m17and 2gs6). ( b ) SDS-PAGE taken for several candidate colorectal carcinoma cell lines, indicating that SW620 COLO320-HSR (as opposed to COLO320-DM, its duplicate line) and COLO741 (later found to be a melanoma line and so not subsequently used here) have negligible native EGFR expression levels compared to positive controls of HCT116, LS180, COLO678 and SW48, shown to have intermediate EGFR expression levels. Note, there is a difference in apparent molecular weight for EGFR between LS180 and COLO678/SW48, most probably due to glycosylation. ( c ) Parental (non GFP) SW620 carcinoma cells show minimal autofluorescence in the green TIRF channel (left panel), while SW620-EGFR-GFP show membrane localization for EGFR-GFP (right panel).

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Transfection, Activation Assay, Generated, SDS Page, Expressing, Molecular Weight, Glycoproteomics, Membrane

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: Confocal microscopy images of fixed cells using GFP, anti-GFP immunofluorescence, and DAPI staining: ( a,b ) non-GFP background cell line SW620; ( c,d ) SW620-EGFR-GFP; ( e ) optical path diagram of bespoke single-molecule TIRF microscope.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Microscopy

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) SDS-PAGE gel indicating generation of Fab nanobody fragments (yellow) from anti-EGF and anti-GFP IgG antibodies (blue), heaving (orange) and light chains (magenta) indicated with reduced Fc (cyan). ( b ) Kernel density estimation distributions of fluorescent foci intensity values measured in kcounts (i.e. counts x 10 3 ) for single GFP (left panel) for live cell, at the end of the photobleach, before EGF is added compared with in vitro Fab and whole IgG data. TMR molecule data for in vitro EGF-TMR and live cell, at the end of the photobleach, post EGF binding data taken from colocalized EGF-EGFR foci is shown (right panel); inset shows live cell EGF-TMR photobleach steps after EGF has been added, taken from colocalized EGF-EGFR foci, with raw (blue) and Chung-Kennedy filter , (red) traces, mean and s.e.m. indicates (arrows).

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: SDS Page, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) TIRF images of surface-immobilized GFP in vitro using IgG and Fab nanobody conjugation. ( b ) Example step-wise photobleach traces show raw (blue) and output data of an edge-preserving Chung-Kennedy filter , (red), kcounts equivalent to counts on our camera detector x 10 3 . ( c ) Example of two nearby SW620-EGFR-GFP cells showing GFP fluorescence (green) and overlaid tracking output (white) with zoom-ins (inset). ( d ) Example photobleach traces from tracked EGFR-GFP foci which have stoichiometries of several tens of EGFR molecules (upper panel), down to an observed minimum of just two molecules (lower panel), raw and overlaid filtered data shown. ( e ) Distribution of EGFR-GFP foci stoichiometry before EGF activation, showing a modal peak at 6 and mean ~12.8 molecules. Data extracted from N=19 cells, detecting N=1,250 foci tracks, corresponding to mean of ~780 EGFR molecules per cell.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: In Vitro, Conjugation Assay, Preserving, Fluorescence, Activation Assay

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: Random overlapping foci predictions for ( a ) monomeric (blue) and dimeric EGFR (magenta), and a mixed model oligomer model suggested from a previous single-molecule study (red) , all showing poor agreement (R 2 <0) to our experimental observations for stoichiometry distribution (grey). ( b ) Monte Carlo Poisson model using an expected average value of 6 molecules for EGFR foci stoichiometry (green) showing reasonable fit (R 2 =0.4923) to experimental data (grey).

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques:

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Brightfield and TIRF images of SW620-EGFR-GFP after adding EGF (~10 min incubation time point), GFP (green), TMR (red) and overlay images shown (yellow indicates high colocalization). ( b ) % of EGFR foci colocalized to EGF, ( c ) number of EGFR-EGF foci detected per cell (s.d. error bars). ( d ) EGFR-EGF foci stoichiometry (red) and isolated EGFR foci (blue) across all EGF incubation times, mean and s.e.m. indicated (arrows), and ( e ) as a function of incubation time (s.d. error bars). We categorized cells into 6 min interval bins resulting in N = 6-12 cells in each bin. ( f ) Distribution of relative stoichiometry of EGFR:EGF, integer bin widths, peak value at 2:1 indicated (arrow). Data extracted from a total of N = 119 cells.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Incubation, Isolation

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Two examples of cells taken ~10 min after the addition of EGF: brightfield images (grey), green channel showing EGFR-GFP localization (green), red channel showing EGF-TMR localization (red), and the overlay of green and red channels together (right panels, with yellow indicating regions of high colocalization) are shown here. ( b ) Variation of the EGFR:EGF relative stoichiometry, rendered as kernel density estimations, as a function of incubation time with EGF (shown in 6 min bins). The region corresponding to 2.0 ± 0.5 relative stoichiometry is indicated as a grey rectangle. ( c ) Heatmap (left panel) and histogram (right panel) characterizing ‘false’ colocalization due to cellular autofluorescence in green and red channels.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Incubation

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Variation of mean EGFR-GFP foci stoichiometry, and ( b ) number of EGFR-GFP foci detected per cell. EGFR-EGF (red) and isolated EGFR foci (blue) are indicated for +/- addition of cetuximab and traztuzumab. Errror bars are s.d, number of cells per dataset in the range N =10 - 117.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Isolation

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Distribution of EGFR foci stoichiometry for cells treated with cetuximab or trastuzumab, showing pre (grey) and post EGF addition for EGFR-EGF (red) and isolated EGFR (blue) foci, data collated across 60 min EGF incubation time, mean and s.e.m. indicated (arrows). ( b ) EGFR:EGF relative stoichiometry of EGFR-EGF foci for drug-treated cells (blue) contrasted against no drug treatment (grey). Number of cells per dataset in the range N =10 - 117.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Isolation, Incubation

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Log-log plot for average mean squared displacement for time intervals of 300 ms or less, and ( b ) log-log plot for apparent microscopic diffusion coefficient D with EGFR stoichiometry S, fits shown to Stokes-Einstein model assuming D ~ S -1/2 (dashed lines). ( c ) EGFR-GFP foci width minus the width of a single GFP vs . stoichiometry, and associated histogram, mean and s.e.m. for all datasets combined indicated (arrow). PreEGF incubation (grey, from N=770 foci, taken from N=19 cells) and post EGF incubation for EGFR-EGF (red, from N=1,969 foci, taken from number N=117 cells) and isolated EGFR (blue, from N=1,741 foci, taken from N=117 cells) foci shown, s.e.m. error bars. ( d ) Histograms EGFR-GFP mean foci width minus width of a single GFP. Pre EGF incubation for cells untreated with drugs (grey, from N=1,252 foci, taken from N=19 cells); cetuximab-treated cells post EGF incubation for EGFR-EGF (red, from N=151 foci, taken from N=10 cells) and isolated EGFR (blue, from N=1,253 foci, taken from N=10 cells) foci shown; trastuzumab-treated cells post EGF incubation for EGFR-EGF (red, from N=263 foci, taken from N=27 cells) and isolated EGFR (blue, from N=1,479 foci, taken from N=27 cells) foci shown; s.e.m. error bars.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Diffusion-based Assay, Incubation, Isolation

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Log-log plot for average mean square displacement vs . time interval for all collated EGFR-GFP foci tracks before addition of EGF, putative confinement zone indicated (dashed lines), from number of foci N=770, acquired from number of cells N=19. ( b ) Log-log plots of EGFR-GFP foci diameters minus the width for a single GFP molecule vs . stoichiometry for not colocalized (left panel) and colocalized foci (right panel), showing cells with no cetuximab or trastuzumab treatment (green, N=6,710 foci, N=117 cells), those treated with cetuximab (magenta, N=1,219 foci, N=25 cells), and those treated with trastuzumab (cyan, N=1,607 foci, N=27 cells), with heuristic power law fit (dash lines), s.e.m. error bars.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques:

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: ( a ) Log-log plots for average mean squared displacement for time intervals of 300 ms or less, and ( b ) log-log plots for variation of apparent microscopic diffusion coefficient D with EGFR stoichiometry S, fits shown to Stokes-Einstein model assuming D ~ S -1/2 (dashed lines) for cetuximab-and trastuzumab-treated cells. ( c ) Histogram of mean Stokes diameter, and ( d ) equivalent diameter values extrapolated for EGFR dimeric foci using same datasets as for , s.e.m. error bars.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Diffusion-based Assay

Journal: bioRxiv

Article Title: EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

doi: 10.1101/305292

Figure Lengend Snippet: Schematic illustrating how HER2 and EGFR dimers may be associated following EGFR activation by EGF (left panel) and how further oligomerization may result in local membrane invagination to form hetero receptor ‘platforms’ of several tens ofnm diameter.

Article Snippet: EGFR protein quantification was performed with western blotting, including cell lines with intermediate levels of EGFR expression as positive controls, probing nitrocellulose membranes with anti-EGFR mouse monoclonal antibody (1:1000, clone 1F4, Cell Signalling Technology ® ) and anti-β-tubulin mouse monoclonal antibody (1:1000, Sigma-Aldrich ® ) prepared in TBS-T, 5% milk and incubated overnight at 4oC.

Techniques: Activation Assay, Membrane

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Top, partial amino acid sequence of the human EphA2 receptor showing the TM helix (underlined), preceded by a short extracellular segment, and followed by the start of the juxtamembrane segment. Residue numbers in the sequence of EphA2 are shown. Middle , sequence of the TYPE7 peptide, where the acidic residues introduced are shown in red. Bottom , sequence of the TMJM 563 -EphA2 peptide used in panel D. ( B ) Circular dichroism determination of TYPE7 secondary structure in buffer at pH 8 (grey line), and in the presence of POPC vesicles at pH 8 (dotted blue line) and after acidification to pH 4 (red line). ( C ) TYPE7 binding to POPC vesicles at pH 5 (red) and pH 8 (blue). Lines are fittings to , used to determine the Kp values. Lipid binding was measured using the environmentally-sensitive dye NBD attached to the N t of TYPE7. ( D ) Determination of the pH midpoint (pH 50 ) for the insertion of TYPE7 into POPC vesicles. TYPE7 data is shown in red symbols. Data obtained in vesicles containing the GWALP23 peptide control are shown in grey, and in vesicles containing TMJM 563 -EphA2 in orange. Peptide insertion was monitored by following changes in the NBD spectral center of mass ( ; ). Control OCD experiments showed that TMJM 563 -EphA2 formed a TM helix . The lines correspond to the fitting to the data using and 95% confidence intervals are shown as shaded areas ( n = 6). ( E ) OCD determination of the membrane orientation of TYPE7. Data were obtained in POPC (16:0,18:1-PC, dashed red line) and 22:1,22:1-PC (continuous red line). The theoretical spectra for a perfectly transmembrane (0°, black line) and peripheral (90°, grey line) helix are shown as a reference. ( F ) Cartoon of the different states TYPE7 (blue) adopts, and how TMJM 563 -EphA2 (orange) promotes the TM state of TYPE7. Arrows represent approximate equilibrium conditions found at pH ~6.5. The (+) symbols represent basic residues in the juxtamembrane segment of EphA2.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Sequencing, Residue, Circular Dichroism, Binding Assay, Control, Membrane

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) TYPE7-bodipy FL-X binding to H358 cells at pH 5, 6 and 7. Data at different pH values were normalized to maximum fluorescence. Mean ±S.D., n = 3. Student’s t -test; *p<0.05; **p<0.01 and NS: not significant. ( B ) H358 cells were treated with increasing concentrations of TYPE7 (0.5, 1 and 2 μM) during 24 hr. Cell viability was assessed using the MTS assay. The results indicate that TYPE7 does not cause toxicity to treated cells. Mean ±S.D., n = 3. ( C ) We threaded the sequence of TYPE7 (blue) onto one of the helices of the published dimeric structure of the transmembrane domain of EphA2 (PDB: 2K9Y) (orange). The residues substituted with glutamic acid are shown as spheres on TYPE7 outside the helix interface. The corresponding EphA2 residues are highlighted on the opposite orange helix.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Fluorescence, MTS Assay, Sequencing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) OCD spectrum of TMJM 563 -EphA2 in POPC (16:0,18:1-PC) bilayers. ( B ) HPLC data showing that TMJM 563 -EphA2 does not dimerize using a disulfide bond. Top , chromatogram showing the elution of the TMJM 563 -EphA2 monomer at 26.2 min. Bottom , control experiment where TMJM 563 -EphA2 dimerization was induced by oxidation with 10 mM copper phenanthroline for 3.5 hr. A dimeric peak appears at 20.2 min, which was not observed in the absence of oxidizing agent.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Confocal microscopy shows co-localization of TYPE7 and EphA2. A375 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 0.2 µM TYPE7-Alexa568 (red) for 5 min at room temperature. Cells were fixed and endogenous EphA2 was labeled via immunofluorescence (green). Images were collected using a 63x objective, and insets show images corresponding to the white dashed areas collected with a 100x objective. Scale bars are 20 µm and 5 µm, respectively. ( B ) The Pearson correlation coefficient (r) was calculated for cells incubated with TYPE7 in the absence and presence of EA1. Bar graph shows mean ±S.D. Student’s t -test was performed for 14 – 17 images. *p<0.05, with as effect size of 0.80 standard deviations, n = 2. ( C ) Top, SDS-PAGE showing that TYPE7-DL co-precipitates with endogenous EphA2 when using a polyclonal anti-rabbit EphA2 antibody. Middle , control Western blots of EphA2 immunoprecipitation blotted with mouse anti-EphA2 show that similar amounts of endogenous EphA2 were pulled down in all samples. Total cell lysates blotted with EphA2 and β-actin indicate that similar levels of protein were loaded. Bottom, quantification of the fluorescent bands. Bar graph shows mean ±S.D. as a percentage of maximum intensity. A Mann-Whitney test was performed (*p<0.05), n = 3.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Confocal Microscopy, Incubation, Labeling, Immunofluorescence, SDS Page, Control, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Left , cell migration was measured in the presence and absence of TYPE7 and EA1 using a Boyden cell chamber assay. Representative images are shown. Right , quantification of migrating cells, showing that incubation with TYPE7 reduced A375 cell migration to a similar degree as EA1, with effect sizes of 8.4 and 12.6 standard deviations from control, respectively. N = 3. Cells were treated with an isolated Fc group as a control for the Fc present in EA1. Scale bar is 200 µm ( B–E ), Phosphorylation of Y772 and JMS phosphorylation at Y588 and Y594. A representative Western blot is shown ( B ). Band intensity was quantified for p-Y772 ( C ), p-Y588 ( D ), and p-Y594 ( E ). We found that incubation with TYPE7 increased phosphorylation of Y772 as efficiently as EA1, with effect sizes of 5.1 and 7.7 standard deviations from control, respectively. Mean ±S.D. are shown. n = 5. ( F–H ), Phosphorylation levels of Akt. A representative Western blot is shown ( F ) and band intensity was quantified for p-T308 ( G ) and p-S473 ( H ). Lysates were blotted against total EphA2 to detect total protein levels, and β-actin as a loading control. Student’s t -test was performed to obtain p values (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 and NS, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, Boyden Chamber Assay, Incubation, Control, Isolation, Phospho-proteomics, Western Blot

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Comparison of the sequences of TYPE7 and pHLIP, with acidic residues marked in orange. Experiments were performed in A375 cells ( B ) and H358 cells ( C ). Top panels , cell lysates were blotted with anti-phospho-EphA2 Y772, and EphA2 and anti-β-actin as loading controls; Bottom panels , quantification of p-EphA2 Y772 bands. Cells were treated with Fc, TYPE7 (2 μM), pHLIP (2 μM), or EA1 (0.5 μg/mL). Statistical analysis was performed using a Student’s t -test; *p<0.05, NS = no significant differences. n = 4 – 6 for panel B, and n = 3 for panel C. All experiments were performed at pH 7.4, except pHLIP in panel B, which was performed at pH 4.2 to ensure complete TM helix formation of pHLIP.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 μg/mL), TYPE7 (2 μM) or EA1 (0.5 μg/mL). The cell lysates were blotted with anti-phospho-EphA2 S897 and anti-β-actin to assess total protein loading. ( B ) EphA2-phospho-S897 quantification of five independent experiments. Statistical analysis was performed by using a Student’s t -test, which indicated no significant differences between samples and controls. ( C ) MTS cell proliferation assay. A375 cells were treated with Fc (0.5 μg/mL), EA1 (3 μg/mL), TYPE7 (2 μM) and TYPE7 +EA1 for 48 hr. No significant differences between Fc control and TYPE7 treated cells were found using a Student’s t -test; **p<0.01. Mean ±S.D., n = 3. ( D–E ) EphA2 expression levels do not change after TYPE7 treatment. Student’s t -test was performed and no significant differences were found between samples. Mean ±S.D., n = 5.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Proliferation Assay, Control, Expressing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 mg/mL), TYPE7 (2 μM), pHLIP (2 μM) or EA1 (0.5 mg/mL). After treatment, cell lysates were incubated overnight with array membranes to detect tyrosine phosphorylation of 49 different RTKs. The three pairs of reference spots used for blot alignment are boxed pink. Boxed RTK are: EphA1 (blue), EphA2 (yellow), HGFR/c-MET (green), EGFR (red) and ErbB3 (orange). ( B ) Bar graph shows mean and standard deviation of selected RTKs. The table on the right shows the identity of all the RTKs. ( C ) pHLIP weakly promotes phosphorylation of ErbB3 and HGFR/c-MET, as TYPE7 does. Since pHLIP does not induce EphA2 phosphorylation at Y772 or affects cell migration , this evidence logically argues against activation of those RTKs being involved in the TYPE7 regulation of these events.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Phospho-proteomics, Standard Deviation, Migration, Activation Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Super-resolution SIM data. H358 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 2 µM TYPE7. Representative images show fluorescence obtained using an anti-EphA2 antibody ( n = 4). Scale bar is 10 µm. Insets magnify areas with clusters, and the scale bars are 5 µm. ( B ) Representative FCS autocorrelation curves for EphA2FL-GFP in control conditions (green) or in the presence of TYPE7 (blue) and EA1 (magenta). Δτ1 and Δτ2 represent the changes in dwell time. ( C–E ) Diffusion coefficient results, containing graphic models describing the EphA2 constructs used. ( C ) Box-whisker plot of measurement of the FCS diffusion coefficient of EphA2FL-GFP. ( D ) Diffusion coefficient of EphA2ΔJ-GFP. ( E ) Diffusion coefficient of Myr-EphA2 ICD-GFP. Diffusion coefficients collected from cells with and without TYPE7 treatment are reported along with EA1 ligand stimulation (orange boxes). The median values are reported next to the box plots. Each data point is the average of five 10 s FCS measurements on one cell. The grey numbers on top of the plots are the total number of cells measured. Criteria for the box, median, quartiles, whiskers and outliers are described elsewhere . One-way ANOVA tests were performed to obtain the p values (****p<0.0001; ns, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Fluorescence, Control, Diffusion-based Assay, Construct, Whisker Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) FCS experiments. Schematic diagram of a FCS experiment. A 488 nm laser beam is focused at the peripheral membrane area of a cultured cell to excite the GFP tag on the diffusive receptors. The emitted photons are collected through the objective and directed to an avalanche photodiode (APD). The fluorescence fluctuation caused by the diffusion of receptors is recorded and transformed into the auto-correlation function. Insert: epi-fluorescence image of DU145 cell expressing GFP-tagged receptors; the red dot represents the position of laser beam. Scale bar is 5 μm. In the auto-correlation curve, τ D and G(0) report on the mobility and the concentration of the diffusive receptors, respectively. ( B ) FCS auto-correlation curves for the three EphA2 constructs. Three curves are shown for each experimental condition. ( C ) Receptor density of EphA2FL-GFP in DU145 cell membranes. Median density value is reported for EphA2FL-GFP and EphA2ΔJ-GFP. Each data point is the average of five 10 s FCS measurements on one cell. 52 cells were measured. ( D ) Representative epi-fluorescence images of cells used for FCS measurements under different conditions of TYPE7 and EA1 treatment. Scale bars are 5 μm.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Membrane, Cell Culture, Fluorescence, Diffusion-based Assay, Transformation Assay, Expressing, Concentration Assay, Construct