Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: REIMS Analysis Predicts Breast Cancer Molecular Markers Including Oncogenic Mutations in PIK3CA (A) Schematic overview of sample preparation for REIMS analysis. (B) Area under the curve (AUC) classification accuracies for ER, PR, HER2 receptor, and triple negative status of 43 breast cancer (BC) cell lines (median intensity of n = 3 biological replicates) following feature selection for phospholipids in the m/z range 600–900, and leave-one-out cross validation. (C) Immunoblot analysis of estrogen inducible protein pS2 and predicted ESR1 expression in ER +ve MCF7 cells following treatment with 0.1% DMSO or indicated concentrations of 4-OHT for 72 h. (D) Unsupervised hierarchical clustering of 872 lipid species detected by REIMS across 43 BC cell lines. (E) Dendrogram of BC cell lines and isogenic MCF10A cells harboring either WT or MUT (E545K or H1047R) PIK3CA. (F) Immunoblot analysis of mature SREBP1 transcription factor expression in nuclear extracts of the MCF10A PIK3CA isogenic panel. (G) Relative exogenous fatty acid uptake in MCF10A PIK3CA WT and MUT cells following serum starvation for 1 h and supplementation with fluorescently labeled dodecanoic acid (n = 5 replicates). (H and I) Unsupervised hierarchical clustering of 9 PIK3CA WT and 9 MUT breast PDX tumors (H) and (I) 5 WT and 7 MUT primary breast tumors. Individual rows in the heatmaps in (D), (H) and (I) correspond to scaled Z score phospholipid intensities (n = 3 biological replicates). Error bars represent ± SEM. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. p values in (C, bottom panel) and (G) were calculated with one-way ANOVA, followed by unpaired, two-tailed Student’s t test with Bonferroni correction.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Sample Prep, Selection, Biomarker Discovery, Western Blot, Expressing, Labeling, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Related to Figure 2 (A) Cell viability of MCF10A PIK3CA MUT cells following treatment with increasing concentrations of rapamycin, torin 1, BYL-719, BKM120, MK2206 or GSK690693 for 72 hours. (B) Unsupervised hierarchical clustering of the median phospholipid intensities of 5 PIK3CA MUT breast cancer cell lines (MCF7, T47D, MDAMB361, MDAMB453 and BT474) treated with 20 nM rapamycin, 100 nM BYL-719 and 150 nM MK2006 for 72 hours. (C) Immunoblot analysis of mTORC1 and mTORC2 signaling in the PIK3CA MUT isogenic panel. Cells were serum and growth-factor starved for 16 hours and subsequently stimulated with 5% horse serum, 20 ng/ml EGF, 0.5 mg/ml hydrocortisone and 10 μg/ml insulin for 30 min. Data in (A) are presented as the mean ± SEM of n = 4 biological replicates and are representative of at least two independent experiments. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01. P value s in (A) were calculated with unpaired, two-tailed Student’s t test.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Western Blot, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet:

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Produced, Virus, Recombinant, Transfection, Protease Inhibitor, Lysis, Mutagenesis, Proliferation Assay, In Situ, Calcium Assay, Reverse Transcription, Kinase Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Proximity Ligation Assay, CRISPR, Control, shRNA, Amplification, Plasmid Preparation, Positive Control, Software, Modification, Targeted Proteomics, Mass Spectrometry, Western Blot

Journal: Disease markers

Article Title: Interferon- γ Stimulates Interleukin-27 Derived from Dendritic Cells to Regulate Th9 Differentiation through STAT1/3 Pathway.

doi: 10.1155/2022/1542112

Figure Lengend Snippet: Figure 8: Characterization of isolated CD4+ T cells stimulated with IFN-γ. CD4+ T cells were extracted from the spleen of C57BL/6 mice and stimulated with 100 ng/mL IFN-γ for 20, 60, or 120 min. (a) Flow cytometry validation of T cell differentiation into Th9 subset with or without IFN-γ stimulation. Cells expressing IL-9 were identified as Th9 cells. (b) qRT-PCR analysis of expression of IL-9 mRNA in unstimulated and IFN-γ-stimulated T cells. (c) ELISA of secreted IL-9 in unstimulated and IFN-γ-stimulated T cells. (d) qRT-PCR of expression of T-bet mRNA in unstimulated and IFN-γ-stimulated (20, 60, or 120 min) T cells. (e) Western blot analysis and quantification of the phosphorylation of STAT1 and STAT3 (relative to total protein content) in unstimulated and IFN-γ-stimulated (20, 60, or 120 min) T cells. Results are presented as mean ± SD (n = 3), ∗p < 0:05. IFN: interferon; IL: interleukin; STAT: signal transducer and activator of transcription.

Article Snippet: Supernatant was discarded, and cells were incubated in flow cytometry buffer containing fluorescein isothiocyanate-conjugated antibodies to CD4 (85-11- 0041-81, eBioscience) for 30min at 4 °C.

Techniques: Isolation, Flow Cytometry, Biomarker Discovery, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Disease markers

Article Title: Interferon- γ Stimulates Interleukin-27 Derived from Dendritic Cells to Regulate Th9 Differentiation through STAT1/3 Pathway.

doi: 10.1155/2022/1542112

Figure Lengend Snippet: Figure 11: Proposed mechanism of IFN-γ-mediated Th9 differentiation through IL-27 release from DCs. When DCs are stimulated by IFN-γ, IL-17 is released, which in turn inhibits the differentiation of CD4+ T cells into a Th9 phenotype and alleviates AA.

Article Snippet: Supernatant was discarded, and cells were incubated in flow cytometry buffer containing fluorescein isothiocyanate-conjugated antibodies to CD4 (85-11- 0041-81, eBioscience) for 30min at 4 °C.

Techniques:

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A and B , images of GPR30 staining intensity level 3 without plasma-membrane staining (GPR30 PM -) ( A ) and with plasma-membrane staining (GPR30 PM +) ( B ). C - G , images of GPR30 total staining (GPR30 TOT ) as negative ( C ), very weak (level 1) ( D ), weak (level 2) ( E ), moderate (level 3) ( F ) and very strong (level 4) ( G ). Bar , 50 μm.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Association between total GPR30 intensity score (GPR30 TOT ), plasma membrane GPR30 (GPR30 PM ) status, and various clinicopathological variables.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Clinical Proteomics, Membrane, Biomarker Discovery, Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: The change in GPR30 intensity (level 0–4) between paired BC and LGL. In relation to the tumor assumed to have developed earlier, the intensity shift in the second tumor is characterized as decreasing, stable or increasing for each tumor pair respectively. The intensity shift was assessed statistically using Wilcoxon matched pairs signed rank test.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Prognostic effect of total GPR30 staining (GPR30 TOT ) and PM-specific GPR30 (GPR30 PM ) staining of BC2 and LGL2 calculated by Cox proportional hazards model with Wald test.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 PM staining of BC2. B , cumulative incidence of BCD in relation to GPR30 PM staining of LGL2. In A and B , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Comparison

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 TOT of BC2 in the whole cohort. B , cumulative incidence of BCD in relation to the five GPR30 staining intensity levels of BC2. C , cumulative incidence of BCD in relation to GPR30 TOT of LGL2. In A - C , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Comparison

Journal: Nature Communications

Article Title: Snail reprograms glucose metabolism by repressing phosphofructokinase PFKP allowing cancer cell survival under metabolic stress

doi: 10.1038/ncomms14374

Figure Lengend Snippet: ( a ) PFK-1 immunoblotting of cell lysates from various human cancer cells. In total, 5 μg of each cell lysate for PFKP and 30 μg of cell lysate for PFKM or PFKL were subjected to immunoblot analysis. The films were developed at the same exposures to compare relative protein abundance of PFK-1 isoforms, and protein loading of each blot was validated with Ponceau stain. The PFK-1 antibody validation using positive control of platelet, muscle and liver tissue are shown in . Cell lines used in this study was authenticated by short tandem repeat profiling as shown . ( b ) Relative transcript (left) and protein (right) abundance of PFKP following knockdown of Snail (shSnail) in breast cancer cells. A total of 20 μg and 5 μg of cell lysates were used to detect endogenous Snail and PFKP, respectively. Representative blots are shown from at least two independent experiments ( a , b ). ( c ) Schematic diagram showing positions of potential Snail-binding canonical E-boxes on the PFKP proximal promoter reporter constructs. Arrow, transcription start site; empty boxes denote E-box mutant. ( d ) Fold increase of reporter activities in combination with wild type or mutated PFKP promoter following Snail shRNA compared with each control shRNA in breast cancer cells. * P <0.05; ** P <0.01 compared with the control. ( e ) Chip-enriched DNA was determined by RT-PCR using specific primers complementary to the promoter regions containing E-box of PFKP (upper) and E-cadherin (lower). Statistical significances ( b , d ) compared with control were denoted as * P <0.05; ** P <0.01 by a two-tailed Student's t -test. Unprocessed original scans of blots are shown in .

Article Snippet: Antibodies against Snail (3,895 s, Cell Signalling, mouse monoclonal, L70G2, 1:2,000), Slug (9585S, Cell Signalling, rabbit, C19G7, 1:1,000), PFKP (sc130227, Santa Cruz, rabbit polyclonal, C-23, 1:1,000), PFKM (HPA002117, Sigma, rabbit polyclonal, 1:1,000), PFKL (sc130226, Santa Cruz, rabbit polyclonal, C-22, 1:1,000), G6PD (sc373887, Santa Cruz, mouse monoclonal, G-6, 1:1,000), Flag (F-3156, Sigma, mouse monoclonal, 1:5,000), HA (12177700, Roche, mouse monoclonal, 12CA5, 1:5,000), O-GlcNAc (MMS-248R, Covance, mouse monoclonal, CTD110.6, 1:1,000) and tubulin (LF-PA0146, AbFrontier, rabbit polyclonal, 1:5,000) were obtained from the commercial vendors.

Techniques: Western Blot, Quantitative Proteomics, Staining, Biomarker Discovery, Positive Control, Knockdown, Binding Assay, Construct, Mutagenesis, shRNA, Control, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Energy Restriction as an Antitumor Target of Thiazolidinediones

doi: 10.1074/jbc.m109.065466

Figure Lengend Snippet: FIGURE 3. TZDs share the ability of 2-DG and glucose starvation to elicit energy restriction-associated cellular responses in LNCaP cells. A, Western blot analysis of the time-dependent effects of 10 M OSU-CG12 vis-a`-vis10mM2-DGandglucosestarvationonvariousmarkersassociatedwithenergyrestriction(inductionof Sirt1 expression, p53 deacetylation, AMPK phosphorylation, and the expression of ER stress indicator GRP78) and with -TrCP-dependent proteolysis (expression of -TrCP, Sp1, and cyclin D1). B, parallel analysis of the mRNA expression levels of Sirt1, GRP78, -TrCP, Sp1, and cyclin D1 by RT-PCR in cells treated with OSU-CG12 and 2-DG as described above. C, Western blot analysis of the effect of TZDs at different doses, relative to 2-DG, resveratrol, and glucose starvation on ER stress and AMPK/mTOR/p70S6K signaling. D, Western blot analysis of the time-dependent effect of 10 M OSU-CG12 versus 10 mM 2-DG on the phosphorylation of eIF-2, a marker associated with energy restriction. TG, troglitazone; CG, ciglitazone; CG12, OSU-CG12; IRE1, inositol-requiring enzyme 1.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies: -catenin, cyclin D1, Wee1, p53, and GFP from Santa Cruz (Santa Cruz, CA); -TrCP from Invitrogen; -actin fromMPBiomedicals (Irvine, CA); rabbit antibodies Myc, PARP, NF B/p105, AR, ER , Ser(P)9-GSK3 , GSK3 , Ser(P)473-Akt, Akt, Thr(P)202/ Tyr(P)204-ERK, ERK, Thr(P)180/Tyr(P)182-p38, p38, Ser(P)/180I B kinase , I B kinase , Thr(P)172-AMPK, AMPK, GRP78, Sirt1, Ac-K382-p53, inositol-requiring enzyme 1 , Ser(P)2448mammalian target of rapamycin (mTOR), mTOR, Thr(P)389- p70S6K, p70S6K, tuberous sclerosis complex (TSC) 2, microtubule-associated protein light chain 3 (LC3), acetyl-lysine (AcK), Ser(P)51-eukaryotic initiation factor (eIF2) , eIF2 , and autophagy-related gene (Atg) 5 from Cell Signaling (Beverly, MA); epidermal growth factor receptor, Sp1, hemagglutinin (HA), and growth arrest and DNA damage-inducible gene (GADD)153 from Santa Cruz; and Atg7 from Abgent (San Diego, CA).

Techniques: Western Blot, Expressing, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Journal of Biological Chemistry

Article Title: Energy Restriction as an Antitumor Target of Thiazolidinediones

doi: 10.1074/jbc.m109.065466

Figure Lengend Snippet: FIGURE5.MechanismsunderlyingOSU-CG12-mediatedinhibitionofglucosemetabolisminLNCaPcells. A, supplemental glucose (20 mg/ml) reversed the transient induction of Sirt1 by OSU-CG12 (CG12) and 2-DG. B, dose-dependent protective effects of supplemental glucose against the induction of PARP cleavage, AMPK activation, and the expression of GRP78 and GADD153 in cells treated with different doses of OSU-CG12 or 2-DG. The ER stress-inducing agent thapsigargin was used as a negative control. C, left panel, dose- and time- dependent effects of OSU-CG12 and resveratrol on [3H]2DG uptake. The data are expressed as the means S.D. (n 3). Right panel, ball-and-stick structures of OSU-CG12 and resveratrol. D, Western blot analysis of the time-dependent suppressive effects of 5 M OSU-CG12 on the phosphorylation of mTOR, p70S6K, and Akt. E, RT-PCR analysis of the dose-dependent effects of OSU-CG12 vis-a`-vis resveratrol on the mRNA levels of hexokinase 2 (HK2) and phosphofructokinase-1 (PFK-1), the first two enzymes in the glycolytic pathway, and fatty acid synthase (FAS) after 24 h of exposure.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies: -catenin, cyclin D1, Wee1, p53, and GFP from Santa Cruz (Santa Cruz, CA); -TrCP from Invitrogen; -actin fromMPBiomedicals (Irvine, CA); rabbit antibodies Myc, PARP, NF B/p105, AR, ER , Ser(P)9-GSK3 , GSK3 , Ser(P)473-Akt, Akt, Thr(P)202/ Tyr(P)204-ERK, ERK, Thr(P)180/Tyr(P)182-p38, p38, Ser(P)/180I B kinase , I B kinase , Thr(P)172-AMPK, AMPK, GRP78, Sirt1, Ac-K382-p53, inositol-requiring enzyme 1 , Ser(P)2448mammalian target of rapamycin (mTOR), mTOR, Thr(P)389- p70S6K, p70S6K, tuberous sclerosis complex (TSC) 2, microtubule-associated protein light chain 3 (LC3), acetyl-lysine (AcK), Ser(P)51-eukaryotic initiation factor (eIF2) , eIF2 , and autophagy-related gene (Atg) 5 from Cell Signaling (Beverly, MA); epidermal growth factor receptor, Sp1, hemagglutinin (HA), and growth arrest and DNA damage-inducible gene (GADD)153 from Santa Cruz; and Atg7 from Abgent (San Diego, CA).

Techniques: Activation Assay, Expressing, Negative Control, Western Blot, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Energy Restriction as an Antitumor Target of Thiazolidinediones

doi: 10.1074/jbc.m109.065466

Figure Lengend Snippet: FIGURE 8. OSU-CG12 induces autophagy by targeting the AMPK/TSC2/mTOR/p70S6K pathway. A, dom- inant-negative or pharmacological inhibition of AMPK blocked OSU-CG12-mediated autophagy but had no effect on ER stress. Left panel, effects of the ectopic expression of the WT versus the K45R kinase-dead, domi- nant-negative (DN) form of AMPK on the ability of OSU-CG12 to modulate the expression levels of p-mTOR, p-p70S6K, -TrCP, and GADD153, and the conversion of GFP-LC3 in GFP-LC3-expressing LNCaP cells. Right panel, parallel analysis of the effects of the AMPK inhibitor compound C. B, shRNA-mediated silencing of Atg5 and Atg7 was used as a positive control to confirm the protective effect of AMPK inhibition on OSU-CG12- induced GFP-LC3 II conversion. C, shRNA-mediated knockdown of TSC2 protected cells from OSU-CG12-in- duced autophagy. Left panel, validation of the effectiveness of shRNA-mediated knockdown of TSC2 by West- ern blot analysis of TSC2 and p-AMPK expression in OSU-CG12-treated cells. Right panel, GFP-LC3-expressing LNCaP cells transfected with scrambled or TSC2 shRNA were exposed to Me2SO (DMSO) or OSU-CG12 for 36 h and then examined by fluorescence microscopy to assess patterns of GFP-LC3 fluorescence. CG12, OSU-CG12.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies: -catenin, cyclin D1, Wee1, p53, and GFP from Santa Cruz (Santa Cruz, CA); -TrCP from Invitrogen; -actin fromMPBiomedicals (Irvine, CA); rabbit antibodies Myc, PARP, NF B/p105, AR, ER , Ser(P)9-GSK3 , GSK3 , Ser(P)473-Akt, Akt, Thr(P)202/ Tyr(P)204-ERK, ERK, Thr(P)180/Tyr(P)182-p38, p38, Ser(P)/180I B kinase , I B kinase , Thr(P)172-AMPK, AMPK, GRP78, Sirt1, Ac-K382-p53, inositol-requiring enzyme 1 , Ser(P)2448mammalian target of rapamycin (mTOR), mTOR, Thr(P)389- p70S6K, p70S6K, tuberous sclerosis complex (TSC) 2, microtubule-associated protein light chain 3 (LC3), acetyl-lysine (AcK), Ser(P)51-eukaryotic initiation factor (eIF2) , eIF2 , and autophagy-related gene (Atg) 5 from Cell Signaling (Beverly, MA); epidermal growth factor receptor, Sp1, hemagglutinin (HA), and growth arrest and DNA damage-inducible gene (GADD)153 from Santa Cruz; and Atg7 from Abgent (San Diego, CA).

Techniques: Inhibition, Expressing, Dominant Negative Mutation, shRNA, Positive Control, Knockdown, Biomarker Discovery, Transfection, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Energy Restriction as an Antitumor Target of Thiazolidinediones

doi: 10.1074/jbc.m109.065466

Figure Lengend Snippet: FIGURE 9. Relative roles of AMPK activation and GADD153 in mediating the effect of OSU-CG12 on apoptosis and viability in LNCaP cells. A, dominant-negative (DN) inhibition of AMPK had no effect on OSU-CG12-induced PARP cleavage (left), but partially protected LNCaP cells from OSU-CG12-mediated sup- pression of cell viability (central) and release of LDH (right). Data are expressed as means S.D. (n 6). B, siRNA-mediated knockdown of GADD153 had no effect on OSU-CG12-induced PARP cleavage or -TrCP induction (left), OSU-CG12-mediated antiproliferative activity (central), or OSU-CG12-mediated LDH release (right). Viability and LDH data are expressed as means S.D. (n 6). C, schematic diagram depicting the mechanism by which TZDs act as ERMAs by perturbing glucose homeostasis, resulting in the hallmark cellular responses, including transient Sirt1 induction, AMPK activation, and ER stress. Our data indicate a mechanistic link between Sirt1 induction and -TrCP protein accumulation, culminating in apoptosis through the protea- somal degradation and transcriptional repression of a series of apoptosis-regulatory proteins. The AMPK acti- vation results in autophagy via the conventional AMPK-TSCs-mTOR-p70S6K pathway. The ER stress signal triggers the up-regulation of sensor proteins, such as GRP78, GADD153 and inositol-requiring enzyme 1 (IRE1), which, might also play a role in down-regulating cell growth.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies: -catenin, cyclin D1, Wee1, p53, and GFP from Santa Cruz (Santa Cruz, CA); -TrCP from Invitrogen; -actin fromMPBiomedicals (Irvine, CA); rabbit antibodies Myc, PARP, NF B/p105, AR, ER , Ser(P)9-GSK3 , GSK3 , Ser(P)473-Akt, Akt, Thr(P)202/ Tyr(P)204-ERK, ERK, Thr(P)180/Tyr(P)182-p38, p38, Ser(P)/180I B kinase , I B kinase , Thr(P)172-AMPK, AMPK, GRP78, Sirt1, Ac-K382-p53, inositol-requiring enzyme 1 , Ser(P)2448mammalian target of rapamycin (mTOR), mTOR, Thr(P)389- p70S6K, p70S6K, tuberous sclerosis complex (TSC) 2, microtubule-associated protein light chain 3 (LC3), acetyl-lysine (AcK), Ser(P)51-eukaryotic initiation factor (eIF2) , eIF2 , and autophagy-related gene (Atg) 5 from Cell Signaling (Beverly, MA); epidermal growth factor receptor, Sp1, hemagglutinin (HA), and growth arrest and DNA damage-inducible gene (GADD)153 from Santa Cruz; and Atg7 from Abgent (San Diego, CA).

Techniques: Activation Assay, Dominant Negative Mutation, Inhibition, Knockdown, Activity Assay

Journal: Frontiers in Endocrinology

Article Title: Mechanistic insights into modified Danggui Buxue Decoction for diabetic retinopathy via integrative analysis

doi: 10.3389/fendo.2025.1648831

Figure Lengend Snippet: Molecular Docking analysis of feature genes and core components of MDBD and validation of GEO datasets. (A) Heatmap of molecular docking analysis of feature genes and core components of MDBD. The horizontal axis is five top feature genes, and the vertical axis is core components. Darker color denotes weaker affinity and lighter color denotes stronger affinity. (B–E) Molecular docking of Beta-sitosterol with CCND1, ERBB2, INSR, and SERPINE1. (F) Differential expression analysis between the control and DR groups. The horizontal axis is feature genes, and the vertical axis is gene expresion level. The data are presented as the mean ± SEM. * P <0.05.

Article Snippet: Beta-sitosterol (HY-N0171A, MedChemExpress, USA) was dissolved in DMSO (D8371, Solarbio, USA).

Techniques: Biomarker Discovery, Quantitative Proteomics, Control

Journal: Frontiers in Endocrinology

Article Title: Mechanistic insights into modified Danggui Buxue Decoction for diabetic retinopathy via integrative analysis

doi: 10.3389/fendo.2025.1648831

Figure Lengend Snippet: Effects of Beta-sitosterol on Hypoxia-Induced MMCs. (A) Cell viability (n=4). The data are presented as the mean ± SEM; *** P < 0.001 vs MOD group. (B) RTqPCR results for ERBB2 gene expression (n=6). The data are presented as the mean ± SEM; *** P < 0.001 vs MOD group. (C, D) Western blotting results of ERBB2 (n=3). The data are presented as the mean ± SEM; *** P < 0.001 vs MOD group. (E) IL-1β levels (n=6). (F) VEGF levels(n=6). (G) ANGPTL6 levels (n=6). Data presented as mean ± SEM. *** p < 0.001 vs MOD group. CON, Control group; MOD, Model group; β-sitosterol, Beta-sitosterol; VEGF, Vascular endothelial growth factor; ANGPTL6, aAngiopoietin-like protein 6; IL-1β, Interleukin-1 beta.

Article Snippet: Beta-sitosterol (HY-N0171A, MedChemExpress, USA) was dissolved in DMSO (D8371, Solarbio, USA).

Techniques: Gene Expression, Western Blot, Control

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A) Immunohistochemistry showing TAGLN2 localized in the germinal center in human secondary lymphoid tissues (left) and Bcl-6 expression, a marker of germinal center B-cells (right) (original magnification, x400). GC, germinal center. The figure is representative of 5 tonsils examined. (B) Double immunofluorescence analysis of TAGLN2 (green) and the B-cell marker CD20 (red), showing coexpression of TAGLN2 and CD20 in the germinal center. Nuclei were stained with DAPI (arrows, original magnification, x400). (C) TAGLN2 mRNA is induced upon BCR stimulation. TAGLN2 mRNA levels were measured in primary human B-cells from six different healthy donors at the indicated time points after IgM+IgG stimulation using qRT-PCR. At each time point, the TAGLN2 mRNA levels in anti-IgM+IgG stimulated cells (stim) are expressed fold change relative to the levels in non-stimulated (non-stim) cells. The mean TAGLN2 mRNA expression ratio was 0.93-fold at 1 h, 1.09-fold at 6 h, and 2.85-fold at 24 h after stimulation, suggesting that TAGLN2 mRNA was induced by 24-h B-cell activation (p<0.05, paired t-test vs. non-stimulated control). Data are presented as the mean ± SEM. *p<0.05.

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Immunohistochemistry, Expressing, Marker, Immunofluorescence, Staining, Quantitative RT-PCR, Activation Assay, Control

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A-B) Double immunofluorescence analysis of TAGLN2 and the B-cell marker CD20 is shown. Nuclei were stained with DAPI. (A) In control lymph nodes, TAGLN2 + B-cells were observed in follicular areas (original magnification, x200). (B) TAGLN2 + B-cells were distributed from the follicular/germinal center (GC) to perifollicular areas in SLE lymphadenopathy (original magnification, x200). HE staining on the left (original magnification, x40). (C) The ratio of TAGLN2+ cells to CD20+ cells in SLE and controls was calculated. Significantly higher numbers of TAGLN2-positive cells were seen in B-cells in lymph nodes associated with SLE (n = 5) compared to those in controls (n = 5). Data are presented as the mean ± SEM. *p<0.05. (D) Right, a few TAGLN2 + CD20 + B-cells (arrows) are seen within B-cell aggregates in the interstitium of lupus nephritis, whereas no TAGLN2 + B-cells are observed in the control kidney sample (Left). (original magnification, x400). (E) Left, TAGLN2 mRNA levels in CD19 + B-cells in healthy donors (HC, n = 6) and SLE patients (n = 9) as determined by qRT-PCR (mean ± SEM, 0.98 ± 0.10 in HC and 0.79 ± 0.04 in SLE) (p>0.05). Right, TAGLN2 mRNA levels in CD19 + CD27 + memory B-cells in healthy donors (HC, n = 11) and SLE patients (n = 14) (the mean of SEM, 1.0 ± 0.11 in HC and 1.2 ± 0.14 in SLE) (p>0.05.).

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Immunofluorescence, Marker, Staining, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Transfection, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of TAGLN2 -knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the TAGLN2 -KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p<0.05. (C) Significant inhibition of CXCL13-dependent TAGLN2 KD Raji cell chemotaxis as compared with negative controls. *p<0.05. RFU, relative fluorescence units. Migratory cells were lysed and quantified using fluorescent dye. The relative quantification was used to determine the change between various samples. Data were normalized by designating one sample in negative controls as equal to 1. Then, the ratiometric results were used to scale all values relative to that sample. (D) The CXCR5 and IgM expression levels were assessed by flow cytometry in TAGLN2 KD and control Raji cells. TAGLN2 KD appeared to have no effect on the surface expression of CXCR5 and IgM. Gray shading indicates isotype control.

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Phospho-proteomics, Membrane, Inhibition, Chemotaxis Assay, Fluorescence, Quantitative Proteomics, Flow Cytometry

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: Pathway analysis of microarray data. Functional grouping of significant, differentially expressed down regulated genes in TAGLN2 siRNA Raji cells versus the control (p <0.01).

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Microarray, Functional Assay, Control, Ligand Binding Assay, Gene Expression, Clinical Proteomics, Membrane, Ubiquitin Proteomics, Transduction

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: Top 30 genes downregulated in TAGLN2 knockdown versus control cells.

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Knockdown, Control, Activity Assay

Journal: PLoS ONE

Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

doi: 10.1371/journal.pone.0184738

Figure Lengend Snippet: (A) Validation of the microarray data regarding the change in expression of specific genes in TAGLN2 -knockdown Raji cells compared to control cells. Expression of the indicated genes was assessed using qRT-PCR and is indicated as fold change relative to that in control Raji cells transfected with scrambled siRNA. Results are the mean ± SEM of triplicate experiments. *p<0.05. (B) CCR6 is colocalized with TAGLN2 + B-cells in GC areas of enlarged follicles in lymph nodes in SLE patients. Double immunofluorescence of CCR6 and TAGLN2 was performed. Nuclei were stained with DAPI. Co-localization of CCR6 and TAGLN2 is indicated in yellow in the merged image (original magnification, x400). GC, germinal center.

Article Snippet: Quantitative real-time PCR (qRT-PCR) reactions were performed in 384-well plate with TaqMan gene probes and primers designed by Life Technologies (CA, USA) for TAGLN2 (assay ID: Hs00761239_s1) and ACTB (assay ID: Hs01060665_gl).

Techniques: Biomarker Discovery, Microarray, Expressing, Knockdown, Control, Quantitative RT-PCR, Transfection, Immunofluorescence, Staining

Journal: Nature metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: Fig. 1 | PEX2 downregulation increases iBA lipolysis and ATGL protein levels in various cell types via reduced poly-ubiquitination. a,b, Levels of glycerol and NEFAs in starvation medium released by iBAs in basal state (n = 4; F = 6.491 (a) and 6.884 (b)). c, IB of HSLSer660ph, HSL, ATGL, CGI-58, PLIN1 and γ-tubulin in iBAs 72 h after Pex2 knockdown (n = 6 in Pex2 and 12 in Nc siRNA, F = 27.6). d, Representative immunofluorescence (IF) result of ATGL in iBAs 72 h after Pex2 knockdown. ATGL in red, LDs in green and nuclei in blue. Scale bar, 20 μm. Experiments were repeated four times. e, IB of ATGL and γ-tubulin in HepG2 cells 48 h after PEX2 knockdown (n = 6 in Pex2 and 10 in Nc siRNA, F = 23.94). f,g, IB of endogenous ATGL or ectopically expressed ATGL–FLAG and γ-tubulin in HEK293T cells 48 h after PEX2 knockdown (n = 6 in Pex2 and 12 in Nc siRNA; F = 17.66 (f) and 20.9 (g)). Results are shown as the mean ± s.e.m. and analysed using ANOVA with Dunnett correction for multiple comparisons between control and other groups. Statistical differences are indicated by exact P values.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used ATGL (1:1000, Cell Signaling:2138S), CGI-58 (1:1000, Proteintech:12201-1-AP), phosphor-HSL(Ser660) (1:1000, Cell Signaling:45804S), PLIN1 (1:1000, Cell Signaling:9349S), HSL (1:1000, Cell Signaling:4107S), γ-tubulin (1:10000, Sigma:T6557), FLAG (1:10000, Sigma:F3165; 1:1000, Cell Signaling:2368S), PEX10 (1:1000, Sigma:WH0005192M1), PEX12 (1:1000, Abcam:ab103456), CAT (1:1000, Cell Signaling:14097S), ACOX1 (1:1000, Abcam:ab184032), HSP90 (1:1000, Cell Signaling:4877S), K48 or K63 linkage specific ubiquitin antibody (1:1000, Cell Signaling:33959), HA (1:1000, Cell signaling:3724S), Myc (1:1000, Sigma:C3956), PMP70 antibody (1;10000, Sigma:SAB4200181), PEX2(1:1000, Thermo:PA5-26187), Calnexin (1:1000, Cell signaling:2433S), EGFP(1:1000, Abcam:ab290), Cysteine sulfenic acid (1:1000, Sigma:ABS30), p-mTORSer2448(1:1000, Cell signaling:2974S), p-S6KThr389(1:1000, Cell signaling:9205S), S6K(1:1000, Cell signaling:9202S), LC3 (1:1000, Cell signaling:2775S), COP1(1:1000, abcam:ab56400), HRPconjugated anti-rabbit IgG (1:10000, Cell signaling: 7074), HRP-conjugated anti-mouse IgG (1:10000, Cell signaling: 7076),Goat antiRabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (1:200, Thermo:A11011),Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200, Thermo:A21202) Validation We validated ATGL,ACOX1 and CAT antibodies in both mouse and human cell lines for WB application via siRNA knockdown.

Techniques: Ubiquitin Proteomics, Knockdown, Immunofluorescence, Control

Journal: Nature metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: Fig. 2 | PEX2 modulates ATGL protein levels via K48-linkage poly-ubiquitination. a, Co-IP conducted in HEK293T whole cell extract (WCE) via FLAG antibody 48 h after expressing PEX2–FLAG. Co-IP was analysed by IB using the indicated antibodies. Experiments were repeated three times. b, Representative images of wild-type ATGL–EGFP, ATGLΔHD–EGFP and ATGL3KR–EGFP distribution in HEK293T cells treated with 400 μM oleic acid (OA). LDs were stained by LipidTOX Deep Red dye and nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. Experiments were repeated three times. c,d, IB of ectopically expressed ATGL3KR–EGFP and ATGLΔHD–EGFP in HEK293T cells 48 h after PEX2 siRNA transfection (n = 8 in PEX2 and 16 in Nc siRNA, F = 3.644 (c); n = 4 in PEX2 and 8 in Nc siRNA, F = 52.67 (d)). e, HEK293T cells were co-transfected by plasmids to express ATGL–FLAG and HA–Ub, followed by siRNA transfection. After 48 h, IP and IB were conducted to detect the ubiquitination pattern. Experiments were repeated three times. f, HEK293T cells were transfected by the ATGL–FLAG plasmid, followed by IP to enrich ATGL–FLAG for poly-ubiquitination type analysis via K48- or K63-linkage poly-ubiquitination antibodies. Experiments were repeated three times. g,h, HEK293T cells were transfected by ATGL–FLAG (g) and ATGLK92–FLAG (h) plasmids, followed by siRNA transfection. After 48 h, IP was conducted to enrich ATGL–FLAG or ATGLK92–FLAG for K48-linkage poly-ubiquitination pattern detection. Experiments were repeated three times. Results are shown as the mean ± s.e.m. and analysed using ANOVA with Dunnett correction for multiple comparisons between control and other groups.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used ATGL (1:1000, Cell Signaling:2138S), CGI-58 (1:1000, Proteintech:12201-1-AP), phosphor-HSL(Ser660) (1:1000, Cell Signaling:45804S), PLIN1 (1:1000, Cell Signaling:9349S), HSL (1:1000, Cell Signaling:4107S), γ-tubulin (1:10000, Sigma:T6557), FLAG (1:10000, Sigma:F3165; 1:1000, Cell Signaling:2368S), PEX10 (1:1000, Sigma:WH0005192M1), PEX12 (1:1000, Abcam:ab103456), CAT (1:1000, Cell Signaling:14097S), ACOX1 (1:1000, Abcam:ab184032), HSP90 (1:1000, Cell Signaling:4877S), K48 or K63 linkage specific ubiquitin antibody (1:1000, Cell Signaling:33959), HA (1:1000, Cell signaling:3724S), Myc (1:1000, Sigma:C3956), PMP70 antibody (1;10000, Sigma:SAB4200181), PEX2(1:1000, Thermo:PA5-26187), Calnexin (1:1000, Cell signaling:2433S), EGFP(1:1000, Abcam:ab290), Cysteine sulfenic acid (1:1000, Sigma:ABS30), p-mTORSer2448(1:1000, Cell signaling:2974S), p-S6KThr389(1:1000, Cell signaling:9205S), S6K(1:1000, Cell signaling:9202S), LC3 (1:1000, Cell signaling:2775S), COP1(1:1000, abcam:ab56400), HRPconjugated anti-rabbit IgG (1:10000, Cell signaling: 7074), HRP-conjugated anti-mouse IgG (1:10000, Cell signaling: 7076),Goat antiRabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (1:200, Thermo:A11011),Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200, Thermo:A21202) Validation We validated ATGL,ACOX1 and CAT antibodies in both mouse and human cell lines for WB application via siRNA knockdown.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Expressing, Staining, Transfection, Plasmid Preparation, Control

Journal: Nature metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: Fig. 6 | ATGL levels are regulated by ROS in HepG2 cells and repressed by overloaded FAs. a,b, HepG2 cells were transfected with CAT or ACOX1 siRNA. After 48 h, IB was conducted to analyse protein levels using the indicated antibodies (n = 6 in CAT and 12 in Nc siRNA, F = 30.1 (a); n = 5 in ACOX1 and 8 in Nc siRNA, F = 20.62 (b)). c,d, HepG2 cells were treated with H2O2 or NAC and collected at the indicated time points. IB was conducted to analyse protein levels using the indicated antibodies (n = 4 in H2O2 treatment and 9 in control, F = 9.689 (c); n = 5 in 6 mM and 7 mM NAC, 6 in 5 mM and 8 mM NAC and 9 in control, F = 47.12 (d)). e, iBAs with PEX2–FLAG–EGFP expression were treated with 0.1 μM Iso and collected at the indicated time points. IP and IB were conducted using the indicated antibodies (n = 4, F = 11.01). f, iBAs were transfected with Nc and Pex2 siRNA. After 48 h, cells were stimulated with 0.1 μM Iso for 24 h at the indicated doses. IB was conducted to check protein levels using the indicated antibodies (n = 4). g–i, iBAs were treated with 100 μM of various FAs. After 48 h, cells were collected for IB analysis or lipolysis measurement was performed (n = 5, F = 14.35 (g), 16.51 (h) and 7.892 (i)). Results are shown as the mean ± s.e.m. and analysed using a two-sided Student’s t-test (f) or ANOVA with Dunnett correction for multiple comparisons between control and other groups (a–e,g–i).

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used ATGL (1:1000, Cell Signaling:2138S), CGI-58 (1:1000, Proteintech:12201-1-AP), phosphor-HSL(Ser660) (1:1000, Cell Signaling:45804S), PLIN1 (1:1000, Cell Signaling:9349S), HSL (1:1000, Cell Signaling:4107S), γ-tubulin (1:10000, Sigma:T6557), FLAG (1:10000, Sigma:F3165; 1:1000, Cell Signaling:2368S), PEX10 (1:1000, Sigma:WH0005192M1), PEX12 (1:1000, Abcam:ab103456), CAT (1:1000, Cell Signaling:14097S), ACOX1 (1:1000, Abcam:ab184032), HSP90 (1:1000, Cell Signaling:4877S), K48 or K63 linkage specific ubiquitin antibody (1:1000, Cell Signaling:33959), HA (1:1000, Cell signaling:3724S), Myc (1:1000, Sigma:C3956), PMP70 antibody (1;10000, Sigma:SAB4200181), PEX2(1:1000, Thermo:PA5-26187), Calnexin (1:1000, Cell signaling:2433S), EGFP(1:1000, Abcam:ab290), Cysteine sulfenic acid (1:1000, Sigma:ABS30), p-mTORSer2448(1:1000, Cell signaling:2974S), p-S6KThr389(1:1000, Cell signaling:9205S), S6K(1:1000, Cell signaling:9202S), LC3 (1:1000, Cell signaling:2775S), COP1(1:1000, abcam:ab56400), HRPconjugated anti-rabbit IgG (1:10000, Cell signaling: 7074), HRP-conjugated anti-mouse IgG (1:10000, Cell signaling: 7076),Goat antiRabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (1:200, Thermo:A11011),Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200, Thermo:A21202) Validation We validated ATGL,ACOX1 and CAT antibodies in both mouse and human cell lines for WB application via siRNA knockdown.

Techniques: Transfection, Control, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: mTOR-related linc-PMB promotes mitochondrial biogenesis via stabilizing SIRT1 mRNA through binding to the HuR protein.

doi: 10.3724/abbs.2024236

Figure Lengend Snippet: Figure 1. Identification of an lncRNA associated with mTOR phosphorylation in HepG2 cells (A) Western blot analysis of HepG2 cells treated with or without rapamycin (20 nM) for 8 h. (B) Hierarchical clustering image of lncRNAs differentially regulated in rapamycin-treated cells compared with vehicle-treated cells. Each row represents an lncRNA. Red: higher expression levels. Green: lower expression levels. (C) Differential expression (fold change ≥2) of 8 lncRNAs in rapamycin-treated cells versus vehicle-treated cells, as determined by microarray analysis. (D) Validation of the differential expression of 4 lncRNAs in rapamycin-treated cells versus vehicle-treated cells via a microarray assay and qRT- PCR. (E) Western blot analysis of HepG2 cells treated with insulin (100 nM) for 20 min followed by 8 h of rapamycin (20 nM) treatment. The relative protein expression of mTORC1 phosphorylation (p-mTOR) was quantified via Image J software (right panel). (F) Gene expression in HepG2 cells treated with or without insulin (100 nM) for 2 h after 8 h of rapamycin (20 nM) or vehicle treatment. PDK4 served as a positive control. (G) Expression levels of linc-PMB in control HepG2 cells and in linc-PMB-knockdown HepG2 cells treated with negative control (NC) or Smart Silencer (Silence) for 24 h, followed by rapamycin (20 nM) treatment for an additional 24 h. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For rapamycin treatment, 70%–80% confluent HepG2 cells were cultured in serum-free medium supplemented with vehicle (0.1% DMSO) or rapamycin (20 nM; Selleck Chemicals, Houston, USA) for 8 h. Microarray analysis lncRNAmicroarray analysis was performed on three paired samples from the rapamycin treatment group and the vehicle control group.

Techniques: Phospho-proteomics, Western Blot, Expressing, Quantitative Proteomics, Microarray, Biomarker Discovery, Quantitative RT-PCR, Software, Gene Expression, Positive Control, Control, Knockdown, Negative Control

Journal: Biomolecules

Article Title: Licochalcone A as a Potential Anti- Toxoplasma Agent: A Target Identification and Pharmacokinetic Study

doi: 10.3390/biom16030410

Figure Lengend Snippet: Screening proteins with anti- Toxoplasma target characteristics via SPR-MS and a multidimensional scoring model. ( A ) Summary table presenting the target protein proteomics results, highlighting a positive correlation between the affinity score and the compound-protein binding capacity. ( B ) Heatmap depicting the scoring results from the target protein proteomics analysis. ( C ) Heatmap illustrating the enrichment abundance from the analysis chip. ( D ) The final comprehensive score shows that MORN1 has the highest score and is predicted to be the most likely key target. As an enzyme protein involved in lipid metabolism. IDH performs well in terms of structural similarity and comparison with known targets but has a weak correlation with the pharmacological mechanism of Lico A. Although ABCB2 may bind Lico A in molecular docking, its criticality and semantic association in parasitic physiology are limited, resulting in a relatively low score. MIC15 ranked fourth in terms of overall score due to its relatively weak structural compatibility. ( E ) The target feature scoring module evaluates the functional similarity between candidate proteins and known drug targets. ( F ) The pharmacological relevance scoring module estimates the semantic correlation between candidate proteins and the known mechanisms of action of Lico A. ( G ) The network-based scoring module assesses the regulatory potential of candidate proteins on the basis of centrality indices within the protein–protein interaction (PPI) network.

Article Snippet: Lico A was acquired from Med Chemexpress (MCE, Monmouth Junction, NJ, USA), item numberHY-N0372.Centrifuge (Thermo Fisher, Waltham, MA, USA),SPR Biochip Analysis System-1 (Berthold, Bad Wildbad, Germany),SPR Unlabeled Interaction Analyzer-2 (PLEXERA LLC, Seattle, WA, USA), Chip microarray printer (BioDot Corporation, Irvine, CA, USA), Gel imaging system (Tanon, Shanghai, China), Fluorescence quantitative PCR (Thermo Lifetech ABI, Waltham, MA, USA), Cell counter (Millipore, Billerica, MA, USA), Protein Stability Analyzer (NanoTemper, Munich, Germany), Flow cytometer (Beckman Coulter, Pasadena, CA, USA), HPLC-MS/MS (AB Sciex, Boston, MA, USA).

Techniques: Protein Binding, Comparison, Functional Assay

Journal: Biomolecules

Article Title: Licochalcone A as a Potential Anti- Toxoplasma Agent: A Target Identification and Pharmacokinetic Study

doi: 10.3390/biom16030410

Figure Lengend Snippet: Validation of Tg MORN1 as the molecular target of Lico A in Toxoplasma gondii tachyzoites. Lico A directly interacts with Tg MORN1 and alters its expression and stability. ( A ) Transcription levels of the Tg MORN1 gene in Toxoplasma gondii before and after dsRNA treatment. ( B ) Rate of Lico A inhibition of Toxoplasma gondii before and after dsRNA treatment. ( C ) Western blot analysis of Tg MORN1 in tachyzoites. Compared to the blank control, treatment with 4 μg/mL Lico A significantly reduced the Tg MORN1 protein level, indicating the downregulation of Tg MORN1 expression (Original images can be found in ). ( D ) Thermal stability of Tg MORN1 as assessed by NanoDSF. The addition of 10 μM Lico A induced a clear shift in the melting curve, reducing the melting temperature (Tm) from 48.1 °C to 44.0 °C, demonstrating the direct interaction of Lico A with Tg MORN1 and decreased protein stability (** significant at p < 0.01; **** significant at p < 0.001).

Article Snippet: Lico A was acquired from Med Chemexpress (MCE, Monmouth Junction, NJ, USA), item numberHY-N0372.Centrifuge (Thermo Fisher, Waltham, MA, USA),SPR Biochip Analysis System-1 (Berthold, Bad Wildbad, Germany),SPR Unlabeled Interaction Analyzer-2 (PLEXERA LLC, Seattle, WA, USA), Chip microarray printer (BioDot Corporation, Irvine, CA, USA), Gel imaging system (Tanon, Shanghai, China), Fluorescence quantitative PCR (Thermo Lifetech ABI, Waltham, MA, USA), Cell counter (Millipore, Billerica, MA, USA), Protein Stability Analyzer (NanoTemper, Munich, Germany), Flow cytometer (Beckman Coulter, Pasadena, CA, USA), HPLC-MS/MS (AB Sciex, Boston, MA, USA).

Techniques: Biomarker Discovery, Expressing, Inhibition, Western Blot, Control, Nano Differential Scanning Fluorimetry

Journal: Biomolecules

Article Title: Licochalcone A as a Potential Anti- Toxoplasma Agent: A Target Identification and Pharmacokinetic Study

doi: 10.3390/biom16030410

Figure Lengend Snippet: Interaction verification and binding site analysis. ( A ) SPR binding curve illustrating the interaction between Lico A and MORN1 across five logarithmic concentration gradients. ( B ) Illustration of three potential binding regions for Lico A on MORN1 through secondary structure representation and spatial structure depiction. The value in parentheses indicates the binding free energy of the compound at that site. ( C ) Schematic representation of bonding interactions between Lico A and nearby amino acid residues at the binding site, with purple arrows denoting hydrogen bonds, yellow arrows denoting van der Waals forces, and green arrows denoting hydrophobic interactions. Summary table of affinity constants. ( D ) The SPR assessment of affinity for wild-type and five point-mutant variants of MORN1, comparing affinity constants (KD). ( E , F ) Concentration gradient binding curves for licochalcone A with wild-type MORN1 (A) the S168A mutant ( E ) and the D203A mutant ( F ). ( G ) Summary table of affinity constants (* significant at p ≤ 0.01).

Article Snippet: Lico A was acquired from Med Chemexpress (MCE, Monmouth Junction, NJ, USA), item numberHY-N0372.Centrifuge (Thermo Fisher, Waltham, MA, USA),SPR Biochip Analysis System-1 (Berthold, Bad Wildbad, Germany),SPR Unlabeled Interaction Analyzer-2 (PLEXERA LLC, Seattle, WA, USA), Chip microarray printer (BioDot Corporation, Irvine, CA, USA), Gel imaging system (Tanon, Shanghai, China), Fluorescence quantitative PCR (Thermo Lifetech ABI, Waltham, MA, USA), Cell counter (Millipore, Billerica, MA, USA), Protein Stability Analyzer (NanoTemper, Munich, Germany), Flow cytometer (Beckman Coulter, Pasadena, CA, USA), HPLC-MS/MS (AB Sciex, Boston, MA, USA).

Techniques: Binding Assay, Concentration Assay, Mutagenesis

Journal: Biomolecules

Article Title: Licochalcone A as a Potential Anti- Toxoplasma Agent: A Target Identification and Pharmacokinetic Study

doi: 10.3390/biom16030410

Figure Lengend Snippet: Plasma concentration–time curve. The two curves respectively represent the time-dependent changes in drug concentration after a single intraperitoneal injection of 150 mg/kg Lico A and a single oral administration of 300 mg/kg Lico A in female BALB/c mice, demonstrating that the drug has favorable in vivo absorption characteristics.

Article Snippet: Lico A was acquired from Med Chemexpress (MCE, Monmouth Junction, NJ, USA), item numberHY-N0372.Centrifuge (Thermo Fisher, Waltham, MA, USA),SPR Biochip Analysis System-1 (Berthold, Bad Wildbad, Germany),SPR Unlabeled Interaction Analyzer-2 (PLEXERA LLC, Seattle, WA, USA), Chip microarray printer (BioDot Corporation, Irvine, CA, USA), Gel imaging system (Tanon, Shanghai, China), Fluorescence quantitative PCR (Thermo Lifetech ABI, Waltham, MA, USA), Cell counter (Millipore, Billerica, MA, USA), Protein Stability Analyzer (NanoTemper, Munich, Germany), Flow cytometer (Beckman Coulter, Pasadena, CA, USA), HPLC-MS/MS (AB Sciex, Boston, MA, USA).

Techniques: Clinical Proteomics, Concentration Assay, Injection, In Vivo

Journal: Biomolecules

Article Title: Licochalcone A as a Potential Anti- Toxoplasma Agent: A Target Identification and Pharmacokinetic Study

doi: 10.3390/biom16030410

Figure Lengend Snippet: Evaluation of the in vitro activity of the Toxoplasma gondii PRU strain by Lico A. ( A ) Lico A decreased the number of PV and parasite number in PV of Toxoplasma gondii Pru strain after 24 h, 48 h, and 72 h incubation. ( B ) Flow cytometry analysis in Lico A induced death in Toxoplasma gondii . The survival of tachyzoites was analyzed by flow cytometry with treatment of 0 μg/mL, 1 μg/mL, 2 μg/mL, or 4 μg/mL Lico A for 24 h. ( C ) Plaques formed by Toxoplasma gondii -infected cells after Lico A treatment. Lico A (0, 1, 2, or 4 μg/mL) decreased Toxoplasma gondii growth inhibition in Vero cells. ( D ) Changes in the shape and size of Toxoplasma gondii PRU strain bradyzoite cyst after Lico A treatment. Toxoplasma gondii were treated with 2 μg/mL Lico A or without drug as a control for 48 h after infecting Toxoplasma gondii PRU strain tachyzoites (* significant at p ≤ 0.01).

Article Snippet: Lico A was acquired from Med Chemexpress (MCE, Monmouth Junction, NJ, USA), item numberHY-N0372.Centrifuge (Thermo Fisher, Waltham, MA, USA),SPR Biochip Analysis System-1 (Berthold, Bad Wildbad, Germany),SPR Unlabeled Interaction Analyzer-2 (PLEXERA LLC, Seattle, WA, USA), Chip microarray printer (BioDot Corporation, Irvine, CA, USA), Gel imaging system (Tanon, Shanghai, China), Fluorescence quantitative PCR (Thermo Lifetech ABI, Waltham, MA, USA), Cell counter (Millipore, Billerica, MA, USA), Protein Stability Analyzer (NanoTemper, Munich, Germany), Flow cytometer (Beckman Coulter, Pasadena, CA, USA), HPLC-MS/MS (AB Sciex, Boston, MA, USA).

Techniques: In Vitro, Activity Assay, Incubation, Flow Cytometry, Infection, Inhibition, Control

Journal: Cell Death & Disease

Article Title: Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

doi: 10.1038/s41419-023-06337-3

Figure Lengend Snippet: Western Blot (WB) analysis of the stable CRISPR-Cas9 mediated knockout (KO) of HSP90α and HSP90β isoforms in K562 ( A ) and in KCL22 ( B ) cells. Clone (C), empty vector (EV), wild type (WT) and non-targeting (NT) control. Beta-actin (β-actin) served as a loading control. Representative immunoblots are shown from three independent repeats. C Expression of other non-cytosolic HSP90 paralogues (GRP94 and TRAP1), HSR-related proteins (HSP70, HSP40 and HSP27) and HSP90 co-chaperones (AHA1 and CDC37) in HSP90β-KO K562 cells. β-actin served as a loading control. D Expression of HSP70, HSP40 and HSP90α upon siRNA mediated HSP90β knockdown (KD) in K562 cells, analyzed by automated WB (JESS). β-actin served as a loading control. E Bars show average protein quantification measurements of HSP90α, HSP70 and HSP40 levels in HSP90β-KD cells compared to NT control cells. Error bars = SD of three independent replicates; p -values were calculated by unpaired two-tailed student’s t-test. F Images of NSG mice ( n = 5 mice/group) transplanted with luciferase-GFP + HSP90α/β-KO or EV K562 cells on the days depicted outside image panel. G Graph show mean ± SD ( n = 5 mice/group) of the bioluminescence measurements of region of interest (radiance; p/s/cm2/sr) at day 35. Significant reduction in the transplantation of HSP90α-KO as compared to EV control K562 cells, determined by unpaired two-tailed student’s t-test, p = 0.0002. H Kaplan–Meier survival curves showing significantly prolonged overall survival of NSG mice transplanted with HSP90α-KO compared to HSP90β-KO or EV (K562) control cells ( n = 5 mice/group, p = 0.0015, Log-rank Mantel-Cox test).

Article Snippet: BCR-ABL1+ chronic myeloid leukemia (CML) cell lines K562, KCL22 and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line SUPB15 (DSMZ, Braunschweig, Germany) were cultured in RPMI1640 GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10–15% FCS and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, CRISPR, Knock-Out, Plasmid Preparation, Control, Expressing, Knockdown, Two Tailed Test, Luciferase, Transplantation Assay

Journal: Cell Death & Disease

Article Title: Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

doi: 10.1038/s41419-023-06337-3

Figure Lengend Snippet: A Volcano plot obtained from five independent replicates of HSP90α-KO compared to EV control K562 cells showing up- or down-regulated proteins based on MS-based secretomics data applying p -value < 0.05 and log2(FC) < −1 or log2(FC) > 1 as the specificity cutoff criteria. The third replicate of the EV control in the secretome data was omitted from the statistical analysis due to its significant deviation from the other four replicates. B Table showing consistently up- or down-regulated proteins from the MS-based proteomic and secretomic analysis in HSP90α-KO cells (upper panel) and HSP90β-KO K562 cells (lower panel). C Synergy maps of PU-H71 and Mitoxantrone combination matrix for two BCR-ABL1 + BCP-ALL patient derived xenograft (PDX) cells, using zero interaction potency (ZIP) method . Visualization was done using SynergyFinder package. WB analysis of CD45 (intracellular domain) and respective inactivating phosphorylation levels of downstream effectors (p-LCK Y505 and p-LYN Y507 ) in HSP90α-KO K562 ( D ) and KCL22 ( E ) cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. F Fluorescence antibody staining (using FACS) validated downregulation of PTPRC (CD45) on the surface of K562 HSP90α-KO cells. G Rescue experiment was performed, in which HSP90α-KO KCL22 cells were transiently transfected with HSP90α-overexpression (OE) construct to re-express the HSP90α isoform.

Article Snippet: BCR-ABL1+ chronic myeloid leukemia (CML) cell lines K562, KCL22 and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line SUPB15 (DSMZ, Braunschweig, Germany) were cultured in RPMI1640 GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10–15% FCS and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Control, Derivative Assay, Phospho-proteomics, Fluorescence, Staining, Transfection, Over Expression, Construct

Journal: Cell Death & Disease

Article Title: Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

doi: 10.1038/s41419-023-06337-3

Figure Lengend Snippet: A Comparative cell viability was measured by luminescent-based ATP-Glo assay after screening HSP90α-KO K562 cells on an ex vivo high throughput drug screening platform, including 93 inhibitors. Average IC 50 values from three independent replicates are depicted in the volcano plots compared to the empty vector (EV) control. Significance was calculated using unpaired t-test. B Synergy map of PU-H71 and THZ1 (CDK7i) combination matrix in a BCR-ABL1 + BCP-ALL PDX sample was generated using Zero Interaction Potency (ZIP) method. Visualization was performed using SynergyFinder package. The experiments were reproduced three times and representative synergy map is shown. WB analysis of K562 ( C ) and KCL22 ( D ) cells following (24 h) treatment with PU-H71, THZ1 alone, or in combination (at depicted concentrations). GAPDH served as a loading control. E Immunofluorescence imaging also confirmed a reduction in the HSP90 and HSP70 levels (in PDX1 cells) upon treatment with a combination of PU-H71 (200 nM) and THZ1 (100 nM), in contrast to the effects of PU-H71 (200 nM) treatment alone. F WB analysis of BCR-ABL1 + BCP-ALL PDX cells after treatment with PU-H71 and THZ1 alone or in combination (at depicted concentrations). GAPDH was used as a loading control. G Schematic depiction of CDK7 inhibition via THZ1 on the expression of HSR-related genes.

Article Snippet: BCR-ABL1+ chronic myeloid leukemia (CML) cell lines K562, KCL22 and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line SUPB15 (DSMZ, Braunschweig, Germany) were cultured in RPMI1640 GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10–15% FCS and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Glo Assay, Ex Vivo, High Throughput Screening Assay, Drug discovery, Plasmid Preparation, Control, Generated, Immunofluorescence, Imaging, Inhibition, Expressing

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Modeling human endometrial decidualization from the interaction between proteome and secretome.

doi: 10.1210/jc.2010-1825

Figure Lengend Snippet: FIG. 2. 2D-DIGE of decidualized (D) vs. control (C) ESCs and validation. Individual proteins of control cells, decidual cells, and a pooled internal standard were labeled with CyDyes, Cy3, Cy5, and Cy2, respectively, and were mixed and separated on a 2D gel using 24 cm pH 3–11 NL (left to right) strips in the first dimension and 10% PAGE-SDS gels in the second dimension. Gels were scanned to obtain images of the overlay of the two dyes (Cy3, Cy5) (A.1, A.2). The same gels, after fluorescence imaging, were silver-stained and scanned (A.3, A.4). The 53 protein spots found to be significantly deregulated in the decidualizated vs. the control samples (P 0.05) are encircled. The proteins identified by in-gel trypsin digestion and MALDI-TOF/TOF are summarized in Table 1. Validation of the DIGE experiments of -actinin, transgelin, cathepsinB, and transglutaminase by Western blot. B, Protein extracts from endometrial samples used in the DIGE experiments; bands correspond to 107-kDa actinin and 23-kDa transgelin. Both significantly reduced in the decidualized vs. the control cells (densitometry values of 4.5 and 6.1, respectively); 38-kDa cathepsin B and 90-kDa transglutaminase proteins were found up-regulated (densitometry values of 1.4 and 1.7, respectively). C, Validation experiments were also performed in a new set of samples which had not been previously analyzed in the DIGE cohort. The results matched the previous ones. GAPDH was used as a control to normalize protein abundance in all the experiments performed. The densitometric results are provided as the normalized values between the indicated markers and GAPDH.

Article Snippet: PVDF membranes were blocked in PBS and 5% dry nonfat milk at room temperature for 1 h. Immunoblotting was performed with primary antibodies; 2.5 g/ml mouse monoclonal transgelin, 2 g/ml mouse monoclonal actinin, 2 g /ml mouse monoclonal transglutaminase2, and0.25 g/mlmousemonoclonal cathepsinB (all of Abcam, Cambridge, UK) at 4 C. Then membranes were incubatedwith the1:2000dilutionof thepolyclonalgoatantirabbit IgG-HRP (DakoCytomation, CA) and the polyclonal rabbit antigoat IgG-HRP (DakoCytomation) at room temperature for 1 h. PVDF membranes were analyzed with the ECL Western Blotting Analysis System (GE Healthcare, Freiburg, Germany) and were photographed using Fujifilm LAS-300.

Techniques: Control, Biomarker Discovery, Labeling, Two-Dimensional Gel Electrophoresis, Fluorescence, Imaging, Staining, Western Blot, Quantitative Proteomics