Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: ( A ) Representative differential interference contrast (DIC) microscopy images of suspended MCF-10A cells showing entotic vacuole or GUVac formation after culture for 24 hr. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm (main panels) or 10 μm (inset). ( B ) Percentage of suspended MCF-10A cells showing entotic vacuole or GUVac formation after 24 hr (n = 772). ( C ) Representative bright-field microscopy images of MCF-10A cells suspended with F-actin cytoskeleton inhibitors. Scale bar: 15 μm ( D ) Percentage of suspended MCF-10A cells showing GUVac formation after exposure to the indicated drugs for the indicated times. dimethyl sulfoxide (DMSO): 0 hr (n = 512), 6 hr (n = 723), 12 hr (n = 690), and 24 hr (n = 690). latrunculin B (LatB): 6 hr (n = 634), 12 hr (n = 693), and 24 hr (n = 428). Cytochalasin D: 6 hr (n = 613), 12 hr (n = 618), and 24 hr (n = 464). Y-27632: 6 hr (n = 448), 12 hr (n = 601), and 24 hr (n = 660). ( E ) Percentage of suspended human primary mammary epithelial cells (HMEpiCs) showing GUVac formation after incubation with DMSO or LatB for the indicated times. DMSO: 6 hr (n = 722), 12 hr (n = 647), 24 hr (n = 417). LatB: 6 hr (n = 1225), 12 hr (n = 1505), and 24 hr (n = 1335). ( F ) Percentage of suspended MCF-10A cells showing GUVac formation after incubation with the indicated drugs for 18 hr. DMSO (n = 1087), LatB (n = 1634), jasplakinolide (n = 2578), Rho inhibitor (n = 839), Y-27632 (n = 1322), EHT 1864 (n = 905), ML 141 (n = 997), nocodazole (n = 1342), and blebbistatin (n = 520). ( G ) Representative DIC microscopy images with phalloidin staining show disruption of the actin cytoskeleton and the GUVac formation in SpvB-expressing cells after suspension culture for 24 hr. Scale bar: 10 μm ( H ) Percentage of GUVac formation in control or SpvB-expressing MCF-10A cells. ( I ) Representative DIC microscopy images after suspension culture of indicated cell lines for 24 hr with LatB. Scale bar: 10 μm ( J ) Representative DIC images of suspended MCF-10A cells treated with DMSO or LatB for the indicated times. White arrowheads indicate the accumulation of vacuoles. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. ( K ) Representative fluorescence and DIC time-lapse images of MCF-10A cells expressing mCherry-H2B and membrane-targeted EGFP obtained at the indicated times after the onset of LatB treatment. Times are presented as hour:minute:second (h:m:s). Scale bars, 10 μm. All quantitative data are means ± SD. The n values represent the total number of cells quantified for two ( D ) or three ( B, E, F, H ) independent experiments. **p<0.01 (two-tailed unpaired t -test) for the indicated comparison ( F, H ) or versus the corresponding value for DMSO ( E ). Figure 1—source data 1. Quantification data corresponding to . Figure 1—source data 2. Quantification data corresponding to . Figure 1—source data 3. Quantification data corresponding to . Figure 1—source data 4. Quantification data corresponding to .

Article Snippet: Reagents were obtained from the following sources and used at the indicated concentrations: LatB (Sigma, 5288, used at 5 μM), cytochalasin D (Sigma, C8273, used at 0.5 μM), jasplakinolide (Tocris, 2792, used at 100 nM), Rho inhibitor I (Cytoskeleton, CT04-A, used at 2 μg/ml), Y-27632 (Sigma, Y0503, used at 20 μM), EHT 1864 (Tocris, 3872, used at 2 μM), ML 141 (Tocris, 4266, used at 10 μM), blebbistatin (Sigma, B0506, used at 50 μM), nocodazole (Sigma, M1404, used at 1 μg/ml), EIPA (Sigma, A3085, used at 50 μM), FITC–dextran (70 kDa) (Sigma, 46945, used at 1 mg/ml), tetramethylrhodamine-dextran (70 kDa) (Invitrogen, D1818, used at 0.1 mg/ml), LysoTracker Red (Invitrogen, L7528, used at 50 nM), CellMask Plasma Membrane Stains Deep Red (Invitrogen, C10046), LY294002 (Sigma, L9908, used at 50 μM), VPS34-IN1 (Cayman, 17392, used at 5 μM), TGX-221 (Cayman, 10007349, used at 50 nM), GDC-0941 (Selleckchem, S1065, used at 100 nM), Dynasore (Sigma, D7693, used at 100 μM), Dynole 34-2 (Tocris, 4222, used at 10 μM), OctMAB (Tocris, 4225, used at 20 μM), MitMAB (Abcam, ab120466, used at 10 μM), FCF (Sigma, C2791, used at 100 μM), Matrigel (Corning, 354230), and recombinant human sFasL (Peprotech, 310-03H).

Techniques: Microscopy, Staining, Incubation, Disruption, Expressing, Suspension, Control, Fluorescence, Membrane, Two Tailed Test, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Estimation of buoyant density of 20S proteasomes after sucrose density gradient centrifugation. ( a ) One μg of commercial 20S proteasome was analyzed by sucrose (15–55%) density gradient centrifugation. After 5 h of high-speed centrifugation, eight fractions (F) were collected, pelleted, and analyzed by gradient SDS-PAGE. The gel was stained with ROTI Blue quick (Carl Roth, Karlsruhe, Germany). Tracks: 1–8—gradient fractions (F). The proteasome subunits were detected predominantly in F2 (1076 g/cm 3 ) and significantly less in F3 (1099 g/cm 3 ). Titration of commercial 20S proteasome (Prot.): 300 ng, 150 ng, 75 ng is shown. ( b ) The buoyant density of fractions and density of proteasome subunits (underlined).

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Gradient Centrifugation, Centrifugation, SDS Page, Staining, Titration

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Release of presumed MVs from HEK293T cells expressing EGFP and quantification of fluorescent vesicles. ( a ) Expression of EGFP in HEK293T cells after 16 h in culture. ( b , c ) “Pre-detachment” stage of large MVs. ( d , e ) Floating extracellular vesicles in cell-free supernatant after filtration through the 0.45 µm and ( f ) through the 0.22 µm membranes, respectively. Bar = 100 µm. Arrows indicate fluorescent particles. ( g ) Estimation of the amount of EVs in 1 µL of cell culture media by flow cytometry. Estimations were performed in triplicates ( h ). Sucrose gradient centrifugation of cell supernatant from HEK293T cells. Each fraction was concentrated 250 times. No proteasome subunits were detected in analyzed gradient fractions. Western blotting was performed using mouse anti-20S alpha 1, 2, 3, 5, 6, 7 proteasome subunits. The limit of the detection by Western blot assay was ~1 ng/band.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Expressing, Filtration, Cell Culture, Flow Cytometry, Gradient Centrifugation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Protein pattern of retroviruses after sucrose gradient centrifugation. Estimation of the CA contents. ( a ) Examined fractions with buoyant density 1.15 g/cm 3 –1.17 g/cm 3 . Track 1—HEK293T (negative control); Tracks 2,3—exogenous JSRV load 7 μL; Track 4—pNLAEKA load 7 μL; Track 5-pNLd load 7 μL; Track 6—endogenous FeLV load 1 μL; Track 7—endogenous RD-114 load 1 μL; Track 8-BSA 250 ng; Track 9—BSA 125 ng; Track 10—exogenous MMTV load 5 μL; Track 11—endogenous RD-114 load 1 μL; Track 12—endogenous FeLV load 1 μL; Track 13—endogenous PERV load 7 μL. CA proteins are given in a frame. ( b ) Serial two-fold dilutions of bovine serum albumin (BSA) were used as a reference for the CA of retroviruses. Centrifugation was performed for 2 h at 110,000 rpm. Numbers below the figures ( a , b ) represented relative band intensity estimated using ImageJ software. Viruses (tracks 2, 4, 6, 7, 10, 11, 12, 13) selected for further proteasome analyses are underlined. Gels were stained with ROTI Blue quick (Carl Roth, Karlsruhe, Germany). M+-color gel markers mixed with ECL markers.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Gradient Centrifugation, Negative Control, Centrifugation, Software, Staining

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Proteasome α and β subunits were detected in sucrose gradient fractions of JSRV, PERV, FeLV, RD-114, and recombinant HIV-1 (pNLd and pNLAEKA). ( a ) Nitrocellulose membrane after SDS-PAGE, protein transfer and Ponceau S staining. The membrane contains gradient fractions F1–F5 of the supernatant from HEK293T cells and fractions F1′–F5′ obtained from supernatant of JSRV-producing cells. CA (arrow) indicates JSRV capsid proteins. Numbers above the image indicate fraction numbers. ( b ) Nitrocellulose membrane containing gradient fractions F1–F8 from the supernatant of PERV-producing cells stained with Ponceau S. CA (arrow) indicates PERV capsid proteins. Numbers above the image indicate fraction numbers. Track 9: PERV gradient fraction F3 grown on FCS-free medium. Track 10—gradient fraction F4 obtained from the supernatant of non-infected HEK293T cells (control). Track 11—20S proteasome 3 ng (control). ( c ) Western blot analysis of gradient fractions F1–F5 from HEK293T cells and gradient fractions F1′–F5′ from JSRV-producingcells with antibodies to 20S proteasome α 1, 2, 3, 5, 6, 7 subunits. ( d ) Western blot analysis of proteasome control and gradient fractions obtained from PERV-producing cells with antibodies to 20S proteasome α 1, 2, 3, 5, 6, 7 subunits. ( e ) Serial dilution (from 50 ng to 3 ng) of commercial 20S proteasome (Enzo, Farmingdale, NY, USA) and detection of proteasome alpha subunits in retroviruses from gradients fractions (F4, F5) using anti-20S proteasome α 1, 2, 3, 5, 6, 7 serum. ( f ) Detection of proteasome beta subunits in fractions of gradient purified retroviruses by Western blotting using anti-β1, anti-β2, and anti-β5 antibodies. Isolated viruses were from fractions F4 and F5. The 12 ng of commercial 20S proteasome (Enzo, Farmingdale, NY, USA) was used as the positive control. M+-Size markers mixed with ECL-size markers. Positions of marker proteins are indicated with bars.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Recombinant, Membrane, SDS Page, Staining, Infection, Control, Western Blot, Serial Dilution, Purification, Isolation, Positive Control, Marker

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Analyses of proteasome activity using Me 4 Bodipy-Ahx 3 -L3-VS cell-permeable proteasome activity probe and fluorogenic peptides. ( a ) Samples were incubated for 1 h with the probe and loaded into the 15% Tris-glycine polyacrylamide gel. The gel was analyzed at the excitation wavelength 480 nm and emission wavelength 530 nm ( left panel ). Fifteen microliters of gradient fractions were loaded. Two microliters of diluted FeLV and RD-114 virus preparations were used. The same gel was stained using ROTI Blue quick (Carl Roth, Karlsruhe, Germany) protein stain ( right panel ). M-Prestained Protein Marker II (10–200 kDa) (Servicebio, Wuhan, China). ( b ) Chymotrypsin-like and caspase-like proteasome activities in viral preparations and gradient fractions. Fractions (F4) of gradient-purified supernatant from non-infected HEK293T cells and HEK293T cells transfected with JSRV, PERV, and pNLAEKA expression clones were used. Chymotrypsin-like and caspase-like proteasome activities were determined using fluorogenic substrates Suc-LLVY-AMC and Z-LLE-AMC, correspondingly. Six microliters of gradient fractions were used. Two microliters of diluted FeLV and one microliter of RD-114 virus preparations were used. Only five microliters of MMTV were used as the amount of sample was limited. Tests were performed in triplicates. The experiments with MMTV were performed in duplicates.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Activity Assay, Incubation, Virus, Staining, Marker, Purification, Infection, Transfection, Expressing, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Putative mechanism of proteasome integration into retroviral particle. Proteasomes might occasionally be integrated into viral particles along with other cellular proteins and nucleic acids.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Retroviral

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Cellular protein targets for functional 20S proteasome in retroviruses.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Functional Assay, Coagulation, Activity Assay

Journal: Cancer biology & therapy

Article Title: Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-β1.

doi: 10.1080/15384047.2020.1824478

Figure Lengend Snippet: Figure 1. Conditioned medium from TAMs upregulates the expression of Gli2 and IGF-II and promotes migration and invasion of Huh-7 cells. (A-B) THP-1 cells were differentiated and polarized into M2 macrophages, and the expression levels of CD68 and CD206 were detected by flow cytometry (A) and quantified (B). (C) After differentiation and polarization, the medium of THP-1 cells was collected, and the concentrations of IL-10 and TGF-β1 were measured by ELISA. (D-E) After treatment with conditioned medium of TAMs (TAMs-CM), the Huh-7 cells were collected, and the levels of TGF-β1, Gli2 and IGF-II were examined by qPCR (D) and western blotting (E). (F) The migration and invasion abilities of Huh-7 cells treated with TAMs-CM were detected by Transwell assay. (G) The migration of Huh-7 cells treated with TAMs- CM was detected by scratch assay. (H) The proliferation of Huh-7 cells treated with TAMs-CM was detected by CCK-8 assay. *** p < .001, ** p < .01, * p < .05.

Article Snippet: In some cases, TGF-β1 inhibitor LY364947 (10, 50, 100 nM), recombinant human IGF-II (20 ng/mL), or ERK1/2 signaling pathway inhibitor PD98059 (50 μM, MedChem Express, Princeton, NJ, USA) were used to treat the Huh-7 cells.

Techniques: Expressing, Migration, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Transwell Assay, Wound Healing Assay, CCK-8 Assay

Journal: Cancer biology & therapy

Article Title: Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-β1.

doi: 10.1080/15384047.2020.1824478

Figure Lengend Snippet: Figure 2. TGF-β1 signaling mediates the effect of TAMs-CM on Gli2 expression and Huh-7 cell migration and invasion. Huh-7 cells were treated with the conditioned medium of TAMs and with different concentrations of the TGF-β1 inhibitor LY364947. (A) The level of Gli2 mRNA was measured by qPCR. (B) The levels of Gli2 and TGF- β1 protein were measured by western blotting. (C-D) The invasion and migration abilities were measured by Transwell assays; the quantification is shown in (D). (E-F) The migration of Huh-7 cells was measured by scratch assay; the quantification is shown in (F). *** p < .001, ** p < .01, * p < .05.

Article Snippet: In some cases, TGF-β1 inhibitor LY364947 (10, 50, 100 nM), recombinant human IGF-II (20 ng/mL), or ERK1/2 signaling pathway inhibitor PD98059 (50 μM, MedChem Express, Princeton, NJ, USA) were used to treat the Huh-7 cells.

Techniques: Expressing, Migration, Western Blot, Wound Healing Assay

Journal: Cancer biology & therapy

Article Title: Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-β1.

doi: 10.1080/15384047.2020.1824478

Figure Lengend Snippet: Figure 6. TAMs promote Huh-7 tumor growth in vivo mediated by TGF-β1. (A-B) TAMs were infected with lentivirus harboring scrambled control shRNA (sh-NC) or shRNA targeting TGF-β1 (sh-TGF-β1). The expression of TGF-β1 was examined by qPCR (A) and western blotting (B). (C) An image of the tumors collected after the mice were sacrificed. (D) The growth rate and weight of tumors in each group. (E) The expression levels of M2 macrophage markers IL-10, Arg-1, CD206 and CD163 in isolated tumors, as detected by qPCR. (F) The expression levels of TGF-β1, Gli2 and IGF-II in tumors detected by qPCR. *** p < .001, ** p < .01, * p < .05.

Article Snippet: In some cases, TGF-β1 inhibitor LY364947 (10, 50, 100 nM), recombinant human IGF-II (20 ng/mL), or ERK1/2 signaling pathway inhibitor PD98059 (50 μM, MedChem Express, Princeton, NJ, USA) were used to treat the Huh-7 cells.

Techniques: In Vivo, Infection, Control, shRNA, Expressing, Western Blot, Isolation

Journal: Blood Advances

Article Title: Unexpected diagnosis of WHIM syndrome in refractory autoimmune cytopenia

doi: 10.1182/bloodadvances.2024013301

Figure Lengend Snippet: Functional assays in CXCR4 variants: enhanced receptor activation and prolonged intracellular signaling. (A) cAMP production was determined in stable NALM6 cells expressing WT CXCR4 (CXCR4 WT ) or CXCR4 variants, after 30 minutes of stimulation in the presence of CXCL12; data represent mean ± standard error of the mean (SEM) of 4 independent experiments. (B) NALM6 cells were serum starved for 4 hours and stimulated with 100 nM rhCXCL12 for 10, 20, or 30 minutes, and ERK1/2 phosphorylation (T202/Y204) was measured by flow cytometry and represented as mean fluorescent intensity (MFI) fold change from baseline. Values represent mean ± SEM of 5 independent experiments. Comparisons are made to WT. ∗ P < .05 (2-tailed unpaired Students t test). (C) Calcium mobilization was determined in WT and CXCR4 mutant NALM6 cells. Measurements were taken every second before and after ligand binding, for 2 minutes. Values represent mean ± SEM of 3 experimental triplicates of 5 independent experiments. (D) Right panel shows the area under the curve (AUC) of calcium mobilization, calculated for each cell line. ∗ P < .05; ∗∗ P < .01 (2-tailed unpaired Student t test). (E) Chemotaxis assay in transwells. Data show the fold of migrated cells to the lower chamber for 4 hours compared with WT. (F) NALM6 cells were serum starved for 4 hours and stimulated with 100 nM rhCXCL12 for 10, 20, or 30 minutes, and AKT phosphorylation (S473) was measured by flow cytometry and represented as MFI fold change from baseline. Values represent mean ± SEM of 5 independent experiments. Comparisons are made with WT. ∗ P < .05; ∗∗ P < .01 (2-tailed unpaired Student t test).

Article Snippet: For this, cells expressing CXCR4 WT or mutants (1 × 10 5 cells per well) were stimulated with 100 nM recombinant human CXCL12 (rhCXCL12; R&D Systems) for 30, 60, 180, and 360 minutes at 37°C, 5% CO 2 .

Techniques: Functional Assay, Activation Assay, Expressing, Phospho-proteomics, Flow Cytometry, Mutagenesis, Ligand Binding Assay, Chemotaxis Assay