Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: List of the human-specific primer sequences used for qPCR analyses.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: Fra-1 regulated p53 signaling pathway activity in cervical cancer cells. (A) mRNA chip analysis in HeLa/vector and HeLa/Fra-1 cells revealed differences in mRNA expression between the two groups. Differentially active signaling pathways were identified by gene ontology analysis. (B) Western blotting analysis of signal transducer and activator of transcription 1 (STAT1), p53, Bcl-2, p38, p21, MDM2, CDK4, and cyclinD1 protein levels in HeLa/vector and HeLa/Fra-1 cells. (C) Real-time polymerase chain reaction (PCR) analysis the mRNA expression levels of STAT1, p53, Bcl-2, p38, p21, MDM2, CDK4, and cyclinD1 in HeLa/vector and HeLa/Fra-1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Protein-Protein interactions, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fra-1 Inhibits Cell Growth and the Warburg Effect in Cervical Cancer Cells via STAT1 Regulation of the p53 Signaling Pathway

doi: 10.3389/fcell.2020.579629

Figure Lengend Snippet: Silencing of signal transducer and activator of transcription 1(STAT1) by small interfering RNA promoted proliferation of cervical cancer cells via activation of the p53 signal pathway. (A) Real-time polymerase chain reaction (PCR) analysis of the silencing effects of three siRNAs for STAT1 in HeLa/Fra-1 cells. (B) Cell viability of HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells according to CCK8 assay. (C) Colony forming ability of HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. (D) In vivo analysis of subcutaneous implanted tumor growth to compare the tumorigenic abilities of HeLa/vector, HeLa/Fra-1, and HeLa/Fra-1/siSTAT1 cells in mice. (E) Western blot analysis of protein expression of STAT1, CDK4, cyclin A, cyclin D1, p53, Bcl-2, c-REL, p38, p21, and MDM2 in HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. (F) Real-time PCR analysis of mRNA expression of STAT1, CDK4, cyclin D1, p53, Bcl-2, p38, p21, and MDM2 in HeLa/Fra-1 and HeLa/Fra-1/siSTAT1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3.

Article Snippet: Then 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene difluoride membrane (HyClone Laboratories, Logan, UT, United States), which was sealed with 5% non-fat milk diluted in PBS-Tween20 for ∼2 h. Primary antibody solution was then added for incubation at 4°C for 12 h. The primary antibodies used were: anti-sirtuin 3 (SIRT3, dilution ratio: 1:500), anti-Mn superoxide dismutase 2 (MnSOD2, dilution ratio: 1:1,000), anti-isocitrate dehydrogenase 2 (IDH2, dilution ratio: 1:500), anti-liver kinase B1 (LKB1, dilution ratio: 1:500), anti-p-AMP-activated protein kinase (AMPK, dilution ratio: 1:1,000), anti-pyruvate kinase muscle isozyme 2 (PKM2, dilution ratio: 1:1,000), anti-phosphofructose kinase 1 (PFK1, dilution ratio: 1:1,000), anti-pyruvate dehydrogenase (PDH, dilution ratio: 1:1,000), anti-FOS like 1, AP-1 transcription factor subunit (FOSL1, Fra-1, Abcam, Cambridge, MA, United States, dilution ratio: 1:500), anti-signal transducer and activator of transcription 1 (STAT1, dilution ratio: 1:500), anti-c-Rel (c-Rel, dilution ratio: 1:1,000), anti-lactate dehydrogenase A (LDHA, dilution ratio: 1:1,000), anti-sirtuin 1 (SIRT1, dilution ratio: 1:500), anti- B-cell lymphoma-2 (Bcl-2, dilution ratio: 1:1,000), anti-NF kappa-B (NF-κB/p65, dilution ratio: 1:1,000, Immunoway Technology, Newark, DE, United States), anti-TP53 (p53, dilution ratio: 1:1,000), anti-cyclin-dependent kinase 4 (CDK4, dilution ratio: 1:1,000), anti-cyclin D1 (dilution ratio: 1:1,000), anti-p21 (dilution ratio: 1:1,000), anti-MAPK14/p38 (dilution ratio: 1:1,000), and anti-MDM2 (dilution ratio: 1:1,000) anti-p16 (dilution ratio: 1:1,000, Boster Technology, Wuhan, China).

Techniques: Small Interfering RNA, Activation Assay, Real-time Polymerase Chain Reaction, CCK-8 Assay, In Vivo, Plasmid Preparation, Western Blot, Expressing

Journal: Nature Communications

Article Title: Prion-induced ferroptosis is facilitated by RAC3

doi: 10.1038/s41467-025-60793-3

Figure Lengend Snippet: A Schematic of a whole-genome CRISPR-activation depletion screen. HT-1080 conditionally expressing PrP C ( PRNP tet ) were transduced with a synergistic activation mediator pooled human library and dCas9-VP64 lentiviruses. PrP C expression was induced for 3 days with doxycycline or vehicle, followed by treatment with 500 nM IKE, resulting in a viability loss of 5–10%. Normalized guide frequencies amplified from viable cells in sensitized PrP C cells were scored against (-)doxycycline control cells to identify enriched or depleted guides. B Fishtail plot of prion-facilitated ferroptosis by differential CRISPRa guide representation. Effect size is determined by the average PRNP tet (+)doxycycline / (−)doxycycline ratio. Depleted guides (Effect size <1) are positive regulators of PrP C -facilitated ferroptosis. C Inverse relationship of RAC3 and PRNP in PrP C OE or RAC3 OE, respectively, by qPCR and Western blot. Relative mRNA expression is shown as mean ± SD of n = 3 technical replicates. D Gene effect characterized by principal component analysis. CRISPR gene effect (individual gene knockout effect on viability and growth, scaled for whole-genome libraries) in 1095 cell lines is displayed in two dimensions. The distance between two genes in the scatter plot can be interpreted to have similar or inverse effects on cellular processes across individual cell lines, relative to all other cell lines. Significance was determined by two-tailed t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human cDNA for PRNP and RAC3 containing was amplified from HT-1080 cDNA and cloned into lentiviral expression vector pLVTHM (Addgene #12247) or pLV (Addgene #39481) with IRES puromycin.

Techniques: CRISPR, Activation Assay, Expressing, Transduction, Amplification, Control, Western Blot, Gene Knockout, Two Tailed Test

Journal: Nature Communications

Article Title: Prion-induced ferroptosis is facilitated by RAC3

doi: 10.1038/s41467-025-60793-3

Figure Lengend Snippet: A Survival of HT-1080 cells treated with RAC-inhibitor EHop-016 (2 μM) and ferroptosis inducer (IKE) rescued by 10 μM α-tocopherol (aToc). B Viability bar graph of RAC3 expressing cells (RAC3 OE) compared to empty vector control with ferroptosis inducer 1.25 μM IKE and 10 μM αToc as a ferroptosis inhibitor. A Western indicates increased expression of RAC3 protein. C Cell survival of PrP C OE, RAC3 OE, and RAC3+PrP c co-expressing cells was detected in freshly infected cell. Cells were treated subsequently with either 2 μM Ferrostatin-1 or DMSO for 3 days. D A flow cytometry histogram depicts the effect of RAC3 OE on induced ROS level via 0.3 μM RSL3 treatment in HT-1080 cells for 3 h measured by DCFH-DA after the indicated time. E Principal component analysis of all metabolomic and lipidomic mass spectrometry features for RAC3 OE cells and cells treated with IKE, compared to control cells. (PC, principal component). F Bar graph illustrates differential expression of 210 annotated phospholipids and lysophospholipids with varying total numbers of double bonds in RAC3 OE cells compared to control cells with an empty vector. Data are representative means ± SD of n = 5 technical replicates. G Differential expression of 210 annotated phospholipids and lysophospholipids with varying total numbers of double bonds in IKE-treated cells compared to vehicle-treated controls. Data are representative means ± SD of n = 5 technical replicates. H Brightfield images of control, RAC3 OE, TGFbeta-1 treated, and RAC3+PrP c OE co-infected cells indicating various levels of roundness (scale bar = 20 µm). I Aspect ratio depicting the effects of TGFbeta-1 treatment, RAC3 OE cells, and RAC3+PrP c co-expressing HT-1080 cells (mean shape, length/width ratio). J Relative expression of epithelial and mesenchymal markers in RAC3 OE and TGFbeta-1 treated cells (3 days) compared to empty vector or DMSO-treated control detected by qPCR. Relative mRNA expression is shown as mean ± SEM of n = 3 technical replicates. Viability data ( A – C ) are representative means ± SEM of n = 3 biological replicates for experiments repeated independently at least three times. The aspect ratio ( I ) are representative means of n = 12 replicates measured by high content microscopy. Significance was determined by two-way ANOVA multiple comparisons with Tukey post-test ( A – C , I ) or two-tailed t -test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human cDNA for PRNP and RAC3 containing was amplified from HT-1080 cDNA and cloned into lentiviral expression vector pLVTHM (Addgene #12247) or pLV (Addgene #39481) with IRES puromycin.

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Infection, Flow Cytometry, Mass Spectrometry, Quantitative Proteomics, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Prion-induced ferroptosis is facilitated by RAC3

doi: 10.1038/s41467-025-60793-3

Figure Lengend Snippet: A Western blotting and densitometry comparing RAC3 protein levels in the brain tissue of mice inoculated with normal brain homogenate (NBH; control) or RML scrapie brain homogenate and sacrificed at 80, 108, and 160 days post inoculation (dpi). Total protein is shown in the lower blot. Data are representative means ± SEM of n = 3 (NBH) or n = 4 (RML) biological replicates. B Western blotting and densitometry comparing RAC3 protein levels in the brain tissue of people who died from sporadic CJD with tissue from patients who died of a non-brain-related condition. Total protein is shown in the lower blot. Data are representative means ± SEM of n = 3 (NBH) or n = 6 (sCJD) biological replicates. C (Left panels) Immunohistochemical detection of prion protein deposits in the cortex of the superior frontal gyrus of Creutzfeldt-Jakob disease (CJD) case (left column) and control case (upper panels, scale bar = 20 µm). A higher magnification shows a punctate synaptic distribution pattern of prion protein deposits typical for both sporadic MM/MV1 CJD cases, whereas no deposits are detectable in the control case (lower panels; scale bar = 200 µm). (Right panels) Immunohistochemical detection of RAC3 in the superior frontal gyrus in a control case (left column) and a case with Creutzfeldt-Jakob disease (CJD, right column). RAC3 is strongly expressed in the neocortical neuropil of the control case (upper left; scale bar = 2 mm), showing a punctate synaptic distribution pattern at higher magnification without expression in cell bodies (lower left; scale bar = 20 µm). In the CJD case, total immunohistochemical signal for RAC3 is strongly reduced in the overview image (upper right; scale bar = 2 mm) compared to the control case. The synaptic distribution pattern is diffuse and unrecognizable at higher magnification (lower right; scale bar = 20 µm). The baseline characteristics of the 6 enrolled patients (3 neuropathological cases and 3 controls) are listed in Table . D A model of prion-induced ferroptosis sensitivity. PrP C expression lowers ER ROS levels via increased GPX8 activity, leading to a relaxed oxidative environment that favors the accumulation of ferroptosis-sensitive PUFA-PL. Oxidative stress facilitated by RAC3 simultaneously depletes these lipids and triggers mesenchymalization, sensitizing to ferroptosis. PrP Sc is speculated to contribute to ferroptosis by increased lipid peroxidation, consistent with observations in infected mice, or by cellular sensing of misfolded PrP, resulting in GPX8 upregulation and sensitivity in the presence of RAC3. Significance was determined two-tailed t -test ( A , B ), P -values = 0.0135 ( A ) and 0.0003 ( B ), respectively. Source data are provided as a Source Data file.

Article Snippet: Human cDNA for PRNP and RAC3 containing was amplified from HT-1080 cDNA and cloned into lentiviral expression vector pLVTHM (Addgene #12247) or pLV (Addgene #39481) with IRES puromycin.

Techniques: Western Blot, Control, Immunohistochemical staining, Expressing, Activity Assay, Infection, Two Tailed Test

Journal: Environmental science. Nano

Article Title: Surface-controlled dissolution rates: a case study of nanoceria in carboxylic acid solutions

doi: 10.1039/C9EN00222G

Figure Lengend Snippet: Dissolution rate coefficients: controls and carboxylic acids.

Article Snippet: Materials The chemicals, their sources, purity, and CAS numbers were adipic acid, TCI, ≥ 99%, 124-04-9; ammonium nitrate, Fisher, ACS grade, 6484-52-2; citric acid monohydrate, Fisher, ACS grade, 5949-29-1; DL-3-hydroxybutyric acid sodium salt, Chem Impex Int’l Inc., 100.30%, 150-83-4 & 306-31-0; DL-malic acid, Alfa Aesar, 98%, 6915-15-7; glutaric acid, Acros organics, 99%,110-94-1; hydrogen peroxide 3% W/W, BDH chemicals, 7722-84-1; horseradish peroxide type II, Sigma, 150-250 U/mg, 9003-99-0; lactic acid, TCI, ≥ 85%, 50-21-5; pimelic acid, Alfa Aesar, 98+%, 111-16-0; sodium acetate, VWR, ACS grade, 127-09-3; sodium azide, Sigma, 99.8%, 26628-22-8; sodium nitrate, BDH chemicals, ACS grade, 7631-99-4; succinic acid, TCI America, ≥ 99%,110-15-6; and tricarballylic acid, Alfa Aesar, 98%, 99-14-9.

Techniques: Dissolution