Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: Whole cell lysate from uninoculated Vero E6, CV-1, A549, Mv1Lu, CRFK, MDCK-NBL-2 and MDCK-SIAT1 cell lines were immunoblotted for endogenous ACE2 expression. Recombinant hACE2 (Sino Biological) was used as a positive control for detection of hACE2. 20 μg of cell lysates or 0.2 ng of recombinant hACE2 protein were loaded. β-actin was also immunoblotted from samples as a loading control.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques: Expressing, Recombinant, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: ACE2 protein sequences from human, rhesus macaque, African green monkey, cat, dog, American mink, mouse, and chicken were aligned using MUSCLE. Residues involved in interaction with SARS-CoV-2 spike protein (based on ref ( – )) are shown using hACE2 numbering, and residues varying from hACE2 are highlighted in yellow. A gap in alignment is indicated with a dash. Percent identity to hACE2 across the entire protein is shown.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques:

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: Cytokine responses in human PBMC induced by various commercial coronavirus spike proteins. ( A ) Rested PBMC were cultured with or without 2.0 μg/mL of raxibacumab (human anti-anthrax PA IgG used as a negative control), MERS S1-Fc, SARS CoV-1 S1-Fc, SARS CoV-2 S1-Fc from Vendor #2 (V#2 S1-Fc) and Vendor #1 (lot 24529-2003, V#1 S1-Fc 2003), and RBD-Fc from Vendor #2 (V#2 RBD-Fc) and Vendor #1 (lot 24530-2003, V#1 RBD-Fc 2003) for 24 hours. ( B ) Rested PBMC were cultured with or without plate-bound streptavidin (STAV) together with or without S1-biotin and RBD-biotin purchased from Vendor #2. The levels of IL-6 and TNFα were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. IL-8 levels were measured using an ELISA kit. The concentrations of the cytokines were calculated based on the standard curves, and the induction of cytokines were presented as stimulation indices. Data shown are statistical results (mean ± SE) generated from 8 ( A ) or 3 ( B ) healthy donors. Statistical analyses were performed by Excel using a two-tailed, Student’s T-test. *, **, *** and **** depict p < 0.05, 0.01, 0.005 and 0.001, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: LPS co-purifying with spike protein reagents induces proinflammatory cytokine production. ( A ) MERS S1-Fc, SARS CoV-1 S1-Fc, S1-Fc from Vendor #2 (V#2 S1-Fc), S1-Fc from Vendor #1 (lot 24056-2002-2 (V#1 S1-Fc 2002) and lot 24529-2003 (V#1 S1-Fc 2003)), RBD-Fc from Vendor #2 (V#2 RBD-Fc) and RBD-Fc from Vendor #1 (lot 25130-2004, V#1 RBD-Fc 2004), and ( B ) V#2 S1-Fc, V#1 S1-Fc 2002 and streptavidin (STAV) before and after treatment of endotoxin removal were assessed for the levels of endotoxin using the LAL Chromogenic Endotoxin Quantitation Kit. The concentrations of endotoxin are presented as EU/mg protein. Data shown are mean ± SE of the results from three independent experiments. ( C ) Time course of S1-Fc-induced cytokine responses. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc and V#1 S1-Fc 2002) for 1, 3, 6 or 24 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were assessed using the CBA human inflammatory cytokine kit and flow cytometric analysis. ( D, E ) Blockade of S1-Fc-induced cytokine response by an LPS inhibitor, polymyxin B. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc) in the presence or absence of polymyxin B for 3 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. Data shown in ( D ) are a representative of the flow cytometric results from three healthy donors and data shown in ( E ) are mean ± SE of the stimulation index derived from three healthy donors, which is calculated using mean fluorescent intensities (MFI) of the cytokines and the formula: cytokine MFI of treated PBMC/cytokine MFI of untreated PBMC. Statistical analyses were performed using a two-tailed, Student’s T-test. * and ** depict p < 0.05 and 0.01, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Quantitation Assay, Cell Culture, Derivative Assay, Two Tailed Test

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Comparison between RBDCoV-ACE2 and a virus neutralization test (VNT). Serum samples (n=16) of pre-pandemic (n=4) and COVID-19 convalescent (n=12) individuals were measured using both assays and analyzed by linear regression. The equation of the dashed regression line is shown next to the graph. VNT results are depicted as half-maximal inhibiting serum dilutions (VNT 50 ), RBDCoV-ACE2 results are shown in percentage inhibition of ACE2 binding. Correlation analysis was performed after Spearman and the correlation coefficient r is shown.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Neutralization, Inhibition, Binding Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between SARS-CoV-2 NeutraLISA and VNT and comparison to RBDCoV-ACE2. (a) Correlation and linear regression between NeutraLISA and VNT results for pre-pandemic (n=4) and COVID-19 infected (n=12) samples. Correlation analyses were performed after Spearman and correlation coefficients r are shown. (b) Descriptive statistics of the (c) correlation between NeutraLISA and RBDCoV-ACE2. One sample from each individual (n=168) was measured using both assays correlation was calculated after Spearman. Samples were classified as being negative (non-neutralizing) if they had a value below 20% (red lines).

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Infection

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: ACE2 binding inhibition varies between RBD mutants. Violin plots showing ACE2 binding inhibition (%) of individual serum samples from 7 to 49 days post PCR (n=50, depicted as dots) against RBD mutants. Black horizontal lines represent medians. Fold-decrease of ACE2 binding inhibition in comparison to wild-type corresponds to the ratio between the medians of wild-type and the respective RBD mutant. VOC-RBDs are shown in blue. Mutations of each RBD mutant are shown in the box above the violin plot.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Mutagenesis

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between anti-RBD IgG MFI signals and ACE2 binding inhibition (%) of serum samples from COVID-19 patients for wild-type and 11 RBD mutants. Regression analysis comparing ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type and all RBD mutants included in the study. Each circle represents one sample (n=168). For longitudinal donors with more than one sample available, the sample closest to 20 days post positive PCR diagnosis was selected. The percentage next to the bracket indicates the proportion of samples with ACE2 binding inhibition ≤ 20% (in orange). Spearman’s correlation coefficient (r) is specified for every correlation.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Longitudinal analysis of ACE2 binding inhibition and anti-RBD IgG levels in Covid-19 patients. Mean ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type RBD against time post positive PCR test for samples (n=149) taken from 1 to 92 days post PCR are shown (a, b). Black dots indicate mean responses with standard deviation indicated by the error bars. The same analysis is then shown for longitudinal samples of selected donors (n=6) for wild-type (c, d) and RBD delta (e, f). For all RBD mutants, mean ACE2 binding inhibition (%) and mean IgG responses (MFI) 1 to 92 days post PCR included in the study is shown (g, h). Each variant is illustrated by a different color according to the figure key.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation of anti-RBD IgG levels and ACE2 binding inhibition with SARS-CoV-2 disease severity. Bar charts showing mean ACE2 binding inhibitions (%) against wild-type and RBD delta are correlated with WHO grades for disease severity for samples 7-49 days post PCR (a, b) and ≥ 50 days post PCR (c, d). Mean anti-RBD WT IgG and anti-RBD delta IgG levels are shown for samples 7-49 days post PCR (e, f) and ≥ 50 days post PCR (g, h). Individual samples are displayed as colored dots, bars indicate the mean of the dataset with error bars representing standard deviation. Number of samples is given below the columns (n). If no samples for a group were available, the column is labeled with “n/a”. WHO grade 1 - ambulatory / no limitations of activities, 2 - ambulatory / limitation of activities, 3 - hospitalized, mild disease / no oxygen therapy, 4 - hospitalized, mild disease / mask or nasal prongs, 6 - hospitalized, severe disease / intubation + mechanical ventilation, 7 - hospitalized, severe disease / ventilation + additional organ support (pressors, RRT, ECMO), 8 – Death. The study did not contain samples of WHO grade 5.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Labeling

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A , B ) The liver tumor tissue microarrays containing 79 tumor tissues (T) and adjacent normal tissues (N) were stained for UBE2F levels, and the representative images of UBE2F staining are shown ( A ). The immunohistochemical (IHC) staining scores of UBE2F in liver tumor tissue microarrays ( A ) were analyzed ( B ). The insets show enlarged images of the red boxes. Scale bars, 120 µm and 60 µm (inset), respectively. Data were presented as median with interquartile range and analyzed by Student’s t test ( n = 79, P = 0.0478). * P < 0.05. ( C ) The Kaplan–Meier survival analysis (using the Mantel–Cox log-rank test) of liver cancer patients with high and low UBE2F expression in ( A ) are shown. The IHC score of UBE2F greater than or equal to 9 is designated as high expression, while the IHC score less than or equal to 4.5 is designated as low expression. ( D , E ) PLC/PRF/5 cells stably expressing shRNA targeting UBE2F were subjected to cell growth assays ( D ) and clonogenic assays ( E ). Representative images of the clonogenic assays are shown (top, E ), and the colony numbers are plotted (bottom, E ). Data were shown as mean ± SEM from three independent experiments, and analyzed by two-way ANOVA ( D ) and one-way ANOVA ( E ), respectively. The P values were 8.3E-14 for siCtrl vs. siUBE2F-1 and 5E-14 for siCtrl vs. siUBE2F-2 ( D ). For ( E ), the P values were 7.95E-5 for both siCtrl vs. siUBE2F-1 and siCtrl vs. siUBE2F-2. *** P < 0.001. ( F ) PLC/PRF/5 cells were transfected with the indicated siRNAs for 48 h and then labeled with 20 μM EdU for 2 h. The cells were subsequently fixed and incubated with Azide 488 before being subjected to flow cytometry. The percentages of proliferating cells were shown as mean ± SEM from three independent experiments and analyzed by one-way ANOVA (siCtrl vs. siUBE2F-1, P = 2.57E-4; siCtrl vs. siUBE2F-2, P = 1.70E-5). *** P < 0.001. ( G , H ) PLC/PRF/5 ( G ) and SK-HEP-1 ( H ) cells were transfected with the indicated siRNAs for 48 h. Cells were then trypsinized and resuspended in PBS, followed by flow cytometry to assess cell size. ( I – K ) Cells were transfected with indicated siRNAs for 72 h, and then stained with anti-LC3 antibody, followed by photography under a confocal fluorescent microscope. Representative images ( I ) and the statistical analysis of the percentages of cells exhibiting LC3-positive puncta ( J , K ) are shown. Data were shown as mean ± SEM from three independent experiments and analyzed by one-way ANOVA. The P values were 0.0109 (siCtrl vs. siUBE2F-1) and 0.0084 (siCtrl vs. siUBE2F-2) for ( J ); 0.0092 (siCtrl vs. siUBE2F-1) and 0.0023 (siCtrl vs. siUBE2F-2) for ( K ). * P < 0.05; ** P < 0.01. Scale bars, 10 µm. ( L ) Cells were transfected with indicated siRNAs for 24 h, and then treated with 10 mM 3-MA or vehicle for an additional 48 h, followed by immunoblotting (IB) analysis using the indicated antibodies (Abs). .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry, Expressing, Stable Transfection, shRNA, Transfection, Labeling, Incubation, Flow Cytometry, Microscopy, Western Blot

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A , B ) Cells stably expressing shRNAs targeting UBE2F were subjected to cell growth assay ( A ) and clonogenic survival assay ( B ). Representative images of the clonogenic assay are shown (top, B ), and colony numbers are plotted (bottom, B ). Data were presented as mean ± SEM from three independent experiments and analyzed by two-way ANOVA ( A ) or one-way ANOVA ( B ), respectively. The P values for the comparisons were as follows: ( A ) shGFP vs. shUBE2F-1 ( P = 5E-14) and shGFP vs. shUBE2F-2 ( P = 5.1E-14). ( B ) shGFP vs. shUBE2F-1 ( P = 2.41E-5) and shGFP vs. shUBE2F-2 ( P = 1.76E-4). *** P < 0.001. ( C – F ) Cells transfected with the indicated siRNA were synchronized in the G1/S phase using 2 mM thymidine to block, followed by releasing with the indicated time periods. Cells were then subjected to FACS analysis ( C ) or IB with the indicated Abs ( F ), and the percentages of cells at the G0/G1 ( D ) and G2/M phases ( E ) are shown. Data are presented as mean ± SEM from three independent experiments and analyzed by two-way ANOVA. The P values for the comparisons were as follows: ( D ) siCtrl vs. siUBE2F-1 ( P = 3.69E-4 for 2 h; 6.19E-5 for 4 h; 0.0235 for 6 h; 0.0033 for 10 h; 0.0012 for 12 h) and siCtrl vs. siUBE2F-2 ( P = 0.007 for 2 h; 6.03E-5 for 4 h; 0.0027 for 6 h; 0.0039 for 10 h; 6.85E-4 for 12 h). ( E ) siCtrl vs. siUBE2F-1 ( P = 3.92E-10 for 4 h, 5.58E-5 for 6 h; 1.25E-4 for 10 h; 0.0025 for 12 h) and siCtrl vs. siUBE2F-2 ( P = 1.47E-8 for 4 h; 1.12E-4 for 6 h; 1.92E-4 for 10 h; 5.82E-4 for 12 h). ** P < 0.01; *** P < 0.001. ( G ) Cells were transfected with indicated siRNAs for 72 h, followed by IB analysis with indicated Abs. ( H ) Cells were transfected with indicated siRNAs for 72 h, autophagosomes were detected by transmission electron microscopy (TEM). The arrows indicated autophagosomes, scale bar, 1 µm. ( I ) Ube2f fl/fl MEF cells were infected with Ad-GFP or Ad-Cre adenovirus for 72 h, followed by IB analysis with indicated Abs. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Stable Transfection, Expressing, Growth Assay, Clonogenic Cell Survival Assay, Clonogenic Assay, Transfection, Blocking Assay, Transmission Assay, Electron Microscopy, Infection

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A ) Cells were transfected with indicated siRNAs for 72 h, followed by IB analysis. ( B ) Cells were transfected with indicated siRNAs for 72 h and then harvested for IB with indicated Abs. ( C , D ) Ube2f fl/fl MEF cells were infected with Ad-GFP or Ad-Cre adenovirus for 72 h. After infection, cells were harvested for IB analysis ( C ), or subjected to serum starvation for 24 h, followed by serum re-supply for indicated time periods before being harvested for IB analysis with indicated Abs ( D ). ( E ) PLC/PRF/5 cells were transfected with siRNA targeting UBE2F or control siRNA (siCtrl) for 48 h, followed by immunoprecipitation (IP) with mTOR Ab, or normal IgG as a negative control. The immunoprecipitated mTOR complexes were then incubated with unphosphorylated purified HA-S6K1 protein, followed by in vitro kinase assay (left). The whole cell extract (WCE) of PLC/PRF/5 cells for IP were subjected to IB analysis with indicated Abs (right). SE short exposure, LE long exposure. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Transfection, Infection, Control, Immunoprecipitation, Negative Control, Incubation, Purification, In Vitro, Kinase Assay

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A ) Sag +/+ and Sag fl/fl MEF cells were infected with Ad-Cre adenovirus for 72 h, and then serum starved for 24 h, followed by serum re-suppy for indicated time periods before being harvested for IB analysis with indicated Abs. ( B – E ) Statistical analysis of the p4E-BP1/4E-BP1 and p-S6K1/S6K1 ratios from Fig. . Data were presented as mean ± SEM from three independent experiments and analyzed by one-way ANOVA. The P values were as follows: ( B ) siCtrl vs. siUBE2F ( P = 2E-4) and siCtrl vs. siSAG ( P = 9E-4). ( C ) siCtrl vs. siUBE2F ( P = 0.0063) and siCtrl vs. siSAG ( P = 0.0042). ( D ) siCtrl vs. siUBE2F ( P = 0.0016) and siCtrl vs. siSAG ( P = 0.0026). ( E ) siCtrl vs. siUBE2F ( P = 3E-4) and siCtrl vs. siSAG ( P = 0.0138). * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) PLC/PRF/5 cells were transfected with indicated siRNAs for 72 h, and then deprived of amino acid for 50 min, followed by re-stimulation with amino acid for 15 min before being harvested for IB analysis with indicated Abs. ( G ) Cells were transfected with indicated siRNAs for 72 h, and then starved of glucose for 6 h, followed by restimulation with glucose for 20 min before being harvested for IB analysis with indicated Abs. ( H ) Hep3B cells transfected with 3 μg of WT-RHEB or the enzymatic-dead mutant UBE2F-C116A were subjected to amino acid or glucose deprivation, followed by refeeding before being harvested for IB analysis with the indicated Abs. ( I , J ) Hep3B and PLC/PRF/5 cells were transfected with indicated siRNAs for 48 h, and then immunostained for mTOR (green) and LAMP2 (red). The statistical analysis of the co-localization of mTOR and LAMP2 was performed by calculating Pearson’s correlation coefficient. Data were presented as mean ± SD from five random fields and analyzed by one-way ANOVA. ns: no significant. Scale bars, 10 µm. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Infection, Transfection, Mutagenesis

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A , B ) Cells were harvested and then subjected to IP with anti-SAG Ab ( A ) or anti-RHEB Ab ( B ), along with normal IgG, followed by IB analysis with indicated Abs. ( C ) A total of 100 ng purified GST-tagged proteins and 50 ng His-SUMO-tagged proteins were mixed in binding buffer and subjected to pull-down using Ni beads (top) or GST beads (bottom), followed by IB analysis with the specified antibodies. ( D ) HEK293 cells were transfected with plasmids expressing FLAG-RHEB and then treated with 1 μM MLN4924 or DMSO as a control for 24 h before being harvested. The cell lysates were then incubated overnight with FLAG beads and subjected to IB using an anti-NEDD8 Ab to detect exogenous RHEB neddylation. ( E ) HEK293 cell lysates were subjected to IP using an anti-NEDD8 Ab, with normal IgG as a control, followed by IB with an anti-RHEB Ab. ( F ) PLC/PRF/5 cells were treated with 1 μM MLN4924 or DMSO as a control for 24 h before being harvested for IP using an anti-RHEB Ab, along with normal IgG as a control. The samples were then analyzed by IB with an anti-NEDD8 Ab to detect endogenous RHEB neddylation. ( G ) HEK293 cells were transfected with the indicated siRNAs for 24 h, followed by transfection with 2 µg HA-RHEB and 1.5 µg HIS-NEDD8 (HIS-N8) for an additional 48 h. The cells were treated with 0.2 µM TAK243 or 1 μM MLN4924 for 12 h, and then lysed under denatured condition. His-tagged neddylated proteins were pulled down by Ni-NTA beads, and then subjected to IB analysis with indicated Abs. ( H ) 100 ng of purified WT-RHEB or RHEB-K169R proteins were added to a reaction mixture containing 200 µM ATP, 0.3 µM NEDD8, 0.025 µM NAE, 0.8 µM UBE2F, and 200 ng of SAG proteins, as indicated. The mixture was continuously mixed for 90 min at 37 °C before being subjected to IB analysis. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Purification, Binding Assay, Transfection, Expressing, Control, Incubation

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A – C ) PLC/PRF/5 cells were simultaneously infected with lentivirus-based shRNA targeting UBE2F or SAG, along with lentivirus expressing WT-RHEB or RHEB-K169R. After puromycin selection, stable cell lines were subjected to cell growth assay ( A ) and clonogenic survival assay ( B , C ). Representative images of colony formation are shown ( B ), and colony numbers are plotted ( C ). Data were shown as mean ± SEM from three independent experiments and analyzed by two-way ANOVA ( A ) or one-way ANOVA ( C ). The P values for the comparisons were as follows: ( A ) shGFP+pLVX vs. shUBE2F+pLVX ( P = 0.0016 for 48 h and 5.10E-6 for 72 h); shGFP+pLVX vs. shSAG+pLVX ( P = 0.0066 for 48 h and 7.37E-7 for 72 h); shUBE2F+pLVX vs. shUBE2F+WT-RHEB ( P = 0.0022 for 48 h and 6.05E-4 for 72 h); shUBE2F+pLVX vs. shUBE2F+RHEB-K169R ( P = 0.9233 for 48 h and 0.9161 for 72 h); shSAG+pLVX vs. shSAG+WT-RHEB ( P = 0.0026 for 48 h and 0.0037 for 72 h); shSAG+pLVX vs. shSAG+RHEB-K169R ( P = 0.9482 for 48 h and 0.9938 for 72 h). ( C ) shGFP+pLVX vs. shUBE2F+pLVX ( P = 1.04E-7); shGFP+pLVX vs. shSAG+pLVX ( P = 6.64E-8); shUBE2F+pLVX vs. shUBE2F+WT-RHEB ( P = 9.29E-4); shUBE2F+pLVX vs. shUBE2F+RHEB-K169R ( P = 0.0767); shSAG+pLVX vs. shSAG+WT-RHEB ( P = 1.45E-4); shSAG+pLVX vs. shSAG+RHEB-K169R ( P = 0.3987). ** P < 0.01; *** P < 0.001; ns no significant. ( D – F ) SK-HEP-1 cells stably overexpressing FLAG-tagged UBE2F or SAG via a lentivirus-based approach were treated with or without 1 µM rapamycin, and then subjected to cell growth assay ( D ) and clonogenic survival assay ( E , F ). Cells were cultured in 2.5% FBS for cell growth assay, representative images of colonies are shown ( E ), and colony numbers are plotted ( F ). Data were shown as mean ± SEM from three independent experiments and analyzed by two-way ANOVA ( D ) or one-way ANOVA ( F ), respectively. The P values for the comparisons were as follows: ( D ) pLVX vs. FLAG-UBE2F ( P = 2E-11); pLVX vs. FLAG-SAG ( P = 1.99E-11); pLVX+Rapamycin vs. FLAG-UBE2F+Rapamycin ( P = 0.9843); pLVX+Rapamycin vs. FLAG-SAG+Rapamycin ( P = 0.8243). ( F ) pLVX vs. FLAG-UBE2F ( P = 0.0196); pLVX vs. FLAG-SAG ( P = 0.0079); pLVX+Rapamycin vs. FLAG-UBE2F+Rapamycin ( P = 0.9334); pLVX+Rapamycin vs. FLAG-SAG+Rapamycin ( P = 0.9680). * P < 0.05; ** P < 0.01; *** P < 0.001; ns: no significant. ( G , H ) Cells were transfected with the indicated siRNA and plasmids for 72 h. The cells were then trypsinized and resuspended in PBS before being subjected to cell size measurement. ( I – L ) PLC/PRF/5 cells were transfected with indicated siRNAs and plasmids for 72 h. Cells were then harvested for IB analysis with indicated Abs ( I , J ), or subjected to immunofluorescent co-staining of LC3 (green) and DAPI (blue) ( K ), and the statistical analysis of the percentages of cells exhibiting LC3-positive puncta are shown ( L ). Data were shown as mean ± SD from five random fields and analyzed by one-way ANOVA. The P values were as follows: siCtrl+Vector vs. siUBE2F+Vector ( P = 0.0025); siCtrl+Vector vs. siSAG+Vector ( P = 5.85E-5); siUBE2F+Vector vs. siUBE2F+RHEB-Q64L ( P = 0.0061); siUBE2F+Vector vs. siUBE2F+RHEB-DM ( P = 0.9672); siSAG+Vector vs. siSAG+RHEB-Q64L ( P = 0.0099); siSAG+Vector vs. siSAG+RHEB-DM ( P = 0.9999). * P < 0.05; ** P < 0.01; ns: no significant. Scale bar, 10 µm. SE short exposure, LE long exposure. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Infection, shRNA, Expressing, Selection, Stable Transfection, Growth Assay, Clonogenic Cell Survival Assay, Cell Culture, Transfection, Staining, Plasmid Preparation

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A ) Hep3B cells stably expressing shRNA and plasmids were subjected to cell growth assay. Data were presented as mean ± SEM from three independent experiments and analyzed by two-way ANOVA. The P values for the various comparisons were as follows: shGFP+pLVX vs. shUBE2F+pLVX ( P = 0.0036 for 48 h and 3.57E-5 for 72 h); shGFP+pLVX vs. shSAG+pLVX ( P = 1.11E-4 for 48 h and 1.16E-7 for 72 h); shUBE2F+pLVX vs. shUBE2F+WT-RHEB ( P = 0.0084 for 48 h and 4.48E-4 for 72 h); shUBE2F+pLVX vs. shUBE2F+RHEB-K169R ( P = 0.9999 for 48 h and 0.9618 for 72 h); shSAG+pLVX vs. shSAG+WT-RHEB ( P = 0.035 for 48 h and 0.0024 for 72 h); shSAG+pLVX vs. shSAG+RHEB-K169R ( P = 0.9999 for both 48 h and 72 h). * P < 0.05; ** P < 0.01; *** P < 0.001; ns: no significant. ( B ) Hep3B cells were infected with lentivirus-based FLAG-UBE2F or FLAG-SAG plasmids, cultured with 2.5% FBS, and then treated with or without 1 μM rapamycin, followed by cell growth assay. Data were presented as mean ± SEM from three independent experiments and analyzed by two-way ANOVA. The P values for the comparisons were as follows: pLVX vs. FLAG-UBE2F ( p = 1.9924E-11); pLVX vs. FLAG-SAG ( P = 1.9904E-11); pLVX+Rapamycin vs. FLAG-UBE2F+Rapamycin ( P = 0.0867); pLVX+Rapamycin vs. FLAG-SAG+Rapamycin ( P = 0.0858). *** P < 0.001; ns: no significant. ( C , D ) Hep3B and SK-HEP-1 cells were infected with the indicated expressing lentivirus and then treated with 100 nM rapamycin for 24 h in 2.5% FBS before being harvesed for IB analysis. ( E – H ) Hep3B and PLC/PRF/5 cells were transfected with the indicated siRNA and plasmids for 48 h, followed by co-staining of LC3 (green) and DAPI (blue) ( E , G ) or IB analysis with the indicated Abs ( F , H ). Scale bar, 10 µm. ( I , J ) Cells were transfected with indicated siRNA and plasmids for 72 h, followed by IB analysis with indicated Abs. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Stable Transfection, Expressing, shRNA, Growth Assay, Infection, Cell Culture, Transfection, Staining

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A ) The livers were harvested from paired littermates with the genotypes of Alb-Cre ; Ube2f +/+ ;Pten fl/fl ( Ube2f + /+;Pten-/- ) and Alb-Cre ; Pten fl/fl ;Ube2f fl/fl ( Ube2f-/-;Pten-/- ) at 3-month old (left) and 6-month old (right), followed by Oil-red-O staining. Scale bars, 40 µm (inset) and 1 mm, respectively. ( B , C ) Analysis of lipid droplet area in liver tissues from paired mouse at the ages of 3 months ( B ) and 6 months ( C ). The area of lipid droplets was quantified using ImageJ from three pairs of mice at each age. For each tissue, data were obtained from five random fields and normalized to the WT mice data. Results are expressed as mean ± SEM, with statistical significance indicated as *** P < 0.001. The P values were: 2.76E-7 for ( B ); and 2.06E-5 for ( C ). ( D – F ) The livers were harvested from paired littermates with the genotypes of Ube2f + /+;Pten-/- ( Ube2f WT) and Ube2f-/-;Pten-/- ( Ube2f KO) at 12-month old ( D ), followed by immunohistochemical staining ( E ) or IB analysis ( F ) with indicated Abs. Scale bar, 40 µm. N normal liver tissues, T tumor tissues. ( G ) Representative image of hepatic cystogenesis (arrow) in Ube2f + /+;Pten-/- and Ube2f-/-;Pten-/- mice at 12-month old. ( H ) H&E staining of liver tissues from ( G ). Scale bars, 1 mm and 40 µm (inset), respectively. ( I ) The livers from two paired Ube2f WT and KO mice at 12-month old were harvested for IB with indicated Abs. ( J ) PLC/PRF/5 cells were transfected with indicated siRNAs for 72 h, followed by IB analysis with indicated Abs. ( K , L ) The liver tumor tissue microarrays containing 81 tumor tissues were stained for UBE2F and pS6 levels. The representative IHC images are shown ( K ), and the correlation analysis of IHC scores between UBE2F and pS6 is shown ( L ). Statistical analysis was conducted using simple linear regression. Scale bars, 125 µm and 40 µm (inset), the insets show enlarged images of the red boxes. ( M ) The Kaplan–Meier survival analysis (using the Mantel–Cox log-rank test) of liver cancer patients with high and low expression of UBE2F and pS6 are shown, both UBE2F and p-S6 IHC scores higher or lower than 6 are referred as UBE2F High/p-S6 High or UBE2F Low/p-S6 Low, respectively. ( N ) A working model for the UBE2F-SAG axis promoting liver steatosis and tumorigenesis via neddylating RHEB to activate mTORC1. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Staining, Immunohistochemical staining, Transfection, Expressing

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: ( A – D ) Liver tissues were isolated from paired Ube2f + /+;Pten-/- (Ube2f WT) and Ube2f-/-;Pten-/- ( Ube2f KO) mice at the ages of 3 and 6 months and then subjected to IB analysis with indicated Abs (A&C) or qRT-PCR analysis (B&D). Data were shown as mean ± SEM and analyzed by Student’s t test. n = 3 for each genotype. The P values were 7E-4 for ( B ) and 0.0199 for ( D ). * P < 0.05; *** P < 0.001. ( E ) The ratios of liver/body weight of Ube2f WT and KO mice at 12-month old were shown as mean ± SEM and analyzed by Student’s t test. P = 0.0471, n = 7 for each genotype; * P < 0.05. ( F ) H&E staining of liver tissues from 12-month-old Ube2f WT and KO mice. Scale bars, 1 mm and 40 µm (inset), respectively. ( G , H ) Quantification of p-S6 ( G ) and p-4Ebp1 ( H ) staining from Fig. . Data were shown as mean ± SD from five random fields and analyzed by Student’s t test. The P values were 0.0014 for ( G ) and 0.0449 for ( H ). * P < 0.05; ** P < 0.01. ( I ) Ck-19 staining of liver sections from 12-month-old Ube2f WT and KO mice. Scale bars, 2 mm and 80 µm (inset), respectively. ( J , K ) Liver tissues from 12-month-old Ube2f WT and KO mice were harvested for IB ( J ) or qRT-PCR analysis ( K ). Data were shown as mean ± SEM and analyzed by Student’s t test. n = 3 for each genotype. The P values were as follows: normal tissue (Ube2f WT vs. KO, P = 1.7E-8) and tumor tissue (Ube2f WT vs. KO , P = 0.0352). * P < 0.05; *** P < 0.001. ( L , M ) SK-HEP-1 cells were transfected with the indicated siRNAs ( L ) or with 3 μg of the indicated plasmids ( M ) for 72 h. The cells were then treated with or without 100 nM rapamycin for 48 h before being harvested for IB with indicated Abs. .

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Isolation, Quantitative RT-PCR, Staining, Transfection

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-UBE2F antibody , Proteintech , Cat #17056-1-AP; RRID: AB_2210295.

Techniques: Mutagenesis, Recombinant, CCK-8 Assay, Software

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence