Journal: Oncotarget

Article Title: RACK1 promotes lung cancer cell growth via an MCM7/RACK1/Akt signaling complex

doi: 10.18632/oncotarget.17120

Figure Lengend Snippet: ( A ) A549 cells were transfected with siRACK1, siCon, GFP-RACK1, or GFP. MCM7 immunoprecipitates were probed for P-Ser, P-Thr, and MCM7 as indicated. ( B ) A549 cells were transfected with siRACK1 or siCon. MCM7 immunoprecipitates were probed for Cdt1, MCM4, MCM6, and MCM7. ( C ) A549 cells were treated with siRACK1, siCon, GFP-RACK1, or GFP as indicated. The chromatin (Chr) and non-chromatin (Non-Chr) fractions of these cells were purified and immunoblotted with anti-MCM7 antibodies. Antibodies against Histone 3 and GADPH were used as internal controls.

Article Snippet: Antibodies for MCM4, MCM6, Cdt1, p27, E2F1, and Histone 3 were from Proteintech Group.

Techniques: Transfection, Purification

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Drug discovery, Infection, Control

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Reverse Transcription, Reflux

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Activity Assay, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Control, Standard Deviation

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Western Blot, Infection, Quantitation Assay, Expressing

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: In Vivo, Injection, Control, Comparison, Infection, Variant Assay, Activity Assay, Virus, Titration, Two Tailed Test

Journal: Animal Cells and Systems

Article Title: Protein DHX38 is a novel inhibitor of protein phosphatase 4

doi: 10.1080/19768354.2015.1074106

Figure Lengend Snippet: Figure 2. DHX38 inhibits PP4-mediated dephosphorylation. (a) Dephosphorylation of RPA2 or KAP1 is promoted in the absence of DHX38, whereas expression of DHX38 significantly disturbs PP4-mediated dephosphorylation. U2OS cells transfected with siRNAs against PP4C or PP4R2, or expression plasmids of PP4R2, PP4C, or DHX38 were irradiated and phospho-RPA2 or phospho-KAP1 was assessed by immunoblot using phospho-specific antibodies. The complete depletion of PP4C and PP4R2 was confirmed by immuno- blot using antibodies against PP4C and PP4R2, and the expression of FH-PP4R2, FH-PP4C, and FH-DHX38 is compatible with their endogenous versions. (b) Expression of DHX38 attenuates pS824-KAP1 turnover after IR. U2OS cells were transfected with DHX38 siRNA or FH-DHX38 plasmid to alter expression. After 72 h, cells were irradiated, fixed at the indicated times and immunostained for pS824-KAP1 (green) and DAPI (blue). Lower Panel: The average pS824-KAP1 signal intensity per nucleus was quantified using ImageJ software. Data are expressed as mean ± SD; n = 3. *P = .0012 (PP4R2), **P = .024 (DHX38), #P = .0027.

Article Snippet: Antibodies used were against PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), PP1γ (Bethyl), PP2AC (Millipore), PP5C (Bethyl), PP6C (Bethyl), WIP1 (Santa Cruz), DHX38 (Proteintech), phospho-RPA2 (Ser33) (Bethyl), RPA2 (Cell Signaling), phospho-KAP1 (Ser824) (Bethyl), pS473-KAP1 (BioLegend), KAP1 (BD Transduction Laboratories), RPA1 (Cell Signaling), Flag-tag (Sigma) and α-tubulin (Sigma).

Techniques: De-Phosphorylation Assay, Expressing, Transfection, Irradiation, Western Blot, Plasmid Preparation, Software

Journal: Animal Cells and Systems

Article Title: Protein DHX38 is a novel inhibitor of protein phosphatase 4

doi: 10.1080/19768354.2015.1074106

Figure Lengend Snippet: Figure 3. DHX38 destabilize PP4R2 (a) Upregulation of PP4R2 by depletion of DHX38. U2OS cells were transfected with siRNAs against DHX38. After 72 h, expression levels of PP4 subunits were detected by immunoblot assay and accessed using ImageJ software (left panel), or mRNA purified from an aliquot of cells was subjected to quantitative real-time PCR (right panel). Data represent average and S.D. of four independent experiments. Compared to control, around 80% of DHX38 was depleted, which was detected by an antibody against DHX38 (left panel). (b) In vitro dephosphorylation assay. PP4C dephosphorylates phospho-KAP1 or phosphor- RPA2 in a dose-dependent manner, but DHX38 has no impact on PP4C-mediated dephosphorylation. PP4C WT and PP4C D82A were purified using the baculoviral system and were serially diluted in the phosphatase reaction. λ phosphatase served as positive control for the reaction. Phosphatase reactions were probed with indicated antibodies.

Article Snippet: Antibodies used were against PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), PP1γ (Bethyl), PP2AC (Millipore), PP5C (Bethyl), PP6C (Bethyl), WIP1 (Santa Cruz), DHX38 (Proteintech), phospho-RPA2 (Ser33) (Bethyl), RPA2 (Cell Signaling), phospho-KAP1 (Ser824) (Bethyl), pS473-KAP1 (BioLegend), KAP1 (BD Transduction Laboratories), RPA1 (Cell Signaling), Flag-tag (Sigma) and α-tubulin (Sigma).

Techniques: Transfection, Expressing, Western Blot, Software, Purification, Real-time Polymerase Chain Reaction, Control, In Vitro, De-Phosphorylation Assay, Positive Control

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: 14 hub genes.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques:

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: RFC4 and GMPS were upregulated in most cancers, especially ESCC. ( A ) Pan-cancer analysis of RFC4. ( B ) Pan-cancer analysis of GMPS. ( C ) Expression of RFC4 in patients with esophageal carcinoma based on histology. ( D ) Expression of GMPS in patients with esophageal carcinoma based on histology. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: Expression analysis of RFC4 and GMPS in ESCC. ( A ) RFC4 mRNA levels in Su Esophagus 2. ( B ) RFC4 mRNA levels in Hu Esophagus 2. ( C ) Comparison of RFC4 across Su and Hu Esophagus. ( D ) GMPS mRNA levels in Su Esophagus 2. ( E ) GMPS mRNA levels in Hu Esophagus 2. ( F ) Comparison of GMPS across Su and Hu Esophagus. ( G , H ) RFC4 and GMPS mRNA levels were detected by RT-qPCR in 46 pairs of ESCC and adjacent normal tissues. ( I ) Protein levels in the three pairs of tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques: Expressing, Comparison, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: Upregulated RFC4 and GMPS are associated with the early diagnosis of esophageal carcinoma and may be the biomarkers for the early diagnosis of esophageal carcinoma. ( A , B ) Expression of RFC4 in patients with esophageal carcinoma based on the stage and nodal metastasis. ( C , D ) Expression of GMPS in patients with esophageal carcinoma based on the stage and nodal metastasis. ( E ) Receiver operating characteristic (ROC) curve analysis of RFC4 and GMPS in esophageal carcinoma ( n = 173). ( F ) Receiver operating characteristic (ROC) curve analysis of RFC4 and GMPS in ESCC ( n = 46). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques: Biomarker Discovery, Expressing

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: Upregulated RFC4 and GMPS levels may be mediated by DNA copy number alterations in ESCC. ( A ) Genetic alteration analysis of RFC4 and GMPS by cBioPortal. ( B , D ) Putative copy number alterations of RFC4 and GMPS in esophageal cancer. ( C , E ) Putative copy number alteration analysis of RFC4 and GMPS based on different T, N, and M stages. ( F , G ) DNA copy number of RFC4 and GMPS in Hu Esophagus 2. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques:

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: Exploration of the mechanism of RFC4 and GMPS based on GSEA and correlation analysis. ( A , B ) KEGG pathway analysis of RFC4 and GMPS based on GSEA. ( C , D ) Correlation coefficient analysis of RFC4 and GMPS in esophageal carcinoma. ( E ) Correlation between RFC4 and GMPS in TCGA. ( F ) Correlation between RFC4 and GMPS in 46 tumor samples. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques:

Journal: Aging (Albany NY)

Article Title: The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis

doi: 10.18632/aging.203520

Figure Lengend Snippet: RFC4 and GMPS expression correlates with tumor-infiltrating immune cells and immune escape in esophageal carcinoma. ( A ) RFC4 expression was positively correlated with the tumor purity (r = 0.287, p < 0.05) but negatively correlated with Dendritic cells (r = −0.193, p < 0.05). ( B ) GMPS expression was positively correlated with the tumor purity (r = 0.217, p < 0.05) but negatively correlated with CD4+ T cells (r = −0.185, p < 0.05) and neutrophils (r =−0.172, p < 0.05). ( C ) A high level of RFC4 accompanied by a high expression of CD4+ T cells ( p = 0.0112), CD 8+ T cells ( p = 0.0164), B cells ( p = 0.00199), dendritic cells ( p = 0.0357) and monocytes ( p = 0.0349) indicated a poor prognosis. When RFC4 was expressed at low levels, a high expression of B cells ( p = 0.0201) was associated with a better prognosis. ( D ) A high level of GMPS accompanied by a high expression of CD 8+ T cells ( p = 0.00804), B cells ( p = 0.0384), dendritic cells ( p = 0.00685) and monocytes ( p = 0.00746) indicated a poor prognosis. When GMPS was expressed at low levels, a high expression of CD 8+ T cells ( p = 0.0442) and dendritic cells ( p = 0.0164) was associated with a better prognosis. p < 0.05 was considered significant.

Article Snippet: The antibodies against RFC4 and GMPS were purchased from Proteintech.

Techniques: Expressing

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: A Immunoblot analysis of stable U2OS cell lines left untreated or incubated with DOX to induce expression of WT or mutant forms of GFP‐FAM111A. B DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse‐labeled with EdU, and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image‐based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6A . C Cells treated as in (B) were pre‐extracted, fixed, and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC ( n > 2,000 cells per condition). D–F Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Fig S6B–D . G Analysis of FAM111A interactors. U2OS/GFP‐FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP), and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/−DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). H U2OS or U2OS/ ΔFAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies. I U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies. J As in (C), except that cells were stained with RFC1 antibody ( n > 2,000 cells per condition). K Quantification of data in (J) for S phase (EdU‐positive) cells (red bars, mean). Data information: Data are representative of at least three (A–F, H–K) and two (G) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Mutagenesis, Labeling, Staining, Cytometry, Immunoprecipitation, Mass Spectrometry, Control, Transfection, Plasmid Preparation

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Validation of FAM111A interactors identified by mass spectrometry (Fig G; ). U2OS/GFP‐FAM111A cell lines treated or not with DOX were subjected to GFP immunoprecipitation (IP) followed by immunoblotting with indicated antibodies. U2OS cells transfected with constructs expressing indicated FLAG‐tagged RFC1 fragments were subjected to FLAG IP followed by immunoblotting with indicated antibodies. U2OS cells transfected with non‐targeting control (CTRL) or FAM111A siRNA were pre‐extracted, fixed, and stained with FAM111A antibody. U2OS cells labeled with EdU were pre‐extracted, fixed, and stained with indicated antibodies. Endogenous FAM111A localizes to nucleoli in G1 phase and G2 phase (EdU‐negative) cells and relocates to nuclear foci in S phase (EdU‐positive) cells. U2OS cells transfected with indicated siRNAs were pre‐extracted, fixed, and stained with FAM111A and RFC1 antibodies. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX. Scale bars, 10 μm. Data information: Data (A–F) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Biomarker Discovery, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Control, Staining, Labeling

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Overview of heterozygous FAM111A mutations found in patients with gracile bone dysplasia or Kenny–Caffey syndrome. Immunoblot analysis of U2OS cell lines left untreated or incubated with DOX to induce expression of the indicated GFP‐FAM111A alleles. U2OS/GFP‐FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP‐FAM111A WT cells used in Figs and (see Fig A). U2OS/GFP‐FAM111A cell lines treated or not with DOX, pulse‐labeled with EdU, and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. Quantification of data in (C) for S phase (EdU‐positive) cells (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6J . As in (C), except that cells were pulse‐labeled with EU. Quantification of EU incorporation in cells in (E) (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6L . U2OS/GFP‐FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC. As in (G), except that cells were stained with RPB1 antibody. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX in the absence or presence of Z‐VAD-FMK. As in (I), using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles. Purified recombinant FLAG‐FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto‐proteolytic activity was analyzed by immunoblotting. GFP IPs from U2OS cell lines expressing GFP‐FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). Data information: Data are representative of at least three (B–K) and two (L) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Labeling, Staining, Purification, Recombinant, Activity Assay, Mutagenesis, Mass Spectrometry

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines conditionally expressing GFP‐FAM111A WT at different levels. Cells in (A) were treated with DOX for 16 h, pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition; A.U., arbitrary units). As in (B), except that cells were treated with DOX for 24 h. Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines expressing WT or patient‐associated GFP‐FAM111A alleles. Cells in (D) were pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Quantification of data in (E) (red bars, mean). Representative images of U2OS/GFP‐FAM111A cell lines that were treated or not with DOX for the indicated times, fixed, and co‐stained with PCNA and RPA2 antibodies. Scale bar, 10 μm. Quantification of data in (G) (gray bars, average; n > 2,000 cells per condition). U2OS/GFP‐FAM111A cell lines treated or not with DOX were stained with γ‐H2AX antibody and analyzed for γ‐H2AX signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6K . As in (I), except that cells were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 signal intensity in S phase cells (gated based on DAPI signal intensity) was analyzed by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6M . As in (I), except that cells were stained with RPB1 antibody and analyzed for RPB1 signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). U2OS cell lines conditionally expressing untagged ectopic FAM111A alleles were treated or not with DOX for 24 h, labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Immunoblot analysis of stable U2OS/FAM111A cell lines transfected or not with FAM111A siRNA targeting the 3′UTR, and subsequently treated or not with DOX to express ectopic untagged FAM111A alleles. Data information: Data (A–M) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Stable Transfection, Expressing, Labeling, Staining, Transfection