Journal:

Article Title: Simultaneous De Novo Identification of Molecules in Chemical Mixtures by Doubly Indirect Covariance NMR Spectroscopy

doi: 10.1021/ja106781r

Figure Lengend Snippet: Doubly indirect covariance spectroscopy of cancer cell line DU145 extract. A) DQF-COSY spectrum, B) 13C-1H HSQC spectrum, C,D) doubly indirect 13C-13C covariance spectrum constructed from parent spectra A, B (see Eq. (2)) before (C) and after (D) removing effects due to 1H overlap, E) carbon-carbon connectivity graphs of mixture components determined from spectrum D. F) Labels a, b, c, d, e, f1 and f2 in Panels E, F indicate ethanol, lactate, taurine, myo-inositol, glutamate, and glutathione reduced, respectively. Graph g in panel E does not match any of the compounds in the database. Myo-inositol displays only 3 nodes (graph d in Panel E) because of 13C chemical shift degeneracy induced by its molecular symmetry.

Article Snippet: An extract from human prostate cancer cell line DU145 (from the American Type Culture Collection (ATCC), www.atcc.org ) was obtained as follows: 14 , 15 the cells were cultured at 37°C in 5% CO 2 incubator in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin.

Techniques: Spectroscopy, Construct

Journal: ACS Chemical Neuroscience

Article Title: Ester Prodrug NLRP3 Inflammasome Inhibitor NT-0796 is Brain Active due to Activation by Local Expression of Carboxylesterase‑1

doi: 10.1021/acschemneuro.5c00843

Figure Lengend Snippet: Evidence of NT-0796 hydrolysis in NHP brain homogenate. (A) NT-0796 (3 μM) was incubated with brain supernatant fraction (#1), CES1 supersomes, or buffer alone. At the indicated times, aliquots of the reaction mixtures were quenched, and concentrations of NDT-19795 were determined by LC-MS/MS analysis and indicated as a function of treatment and time at 37 °C. (B) NHP brain homogenate supernatant fractions #1 and #2, along with CES1 supersomes, were treated with FP-biotin, after which tagged proteins were subsequently captured with streptavidin agarose. After dissociation, the isolates were separated by SDS gel electrophoresis and transferred to nitrocellulose. The blot was stained sequentially with Alexafluor 680-conjugated streptavidin (red) and rabbit anti-CES1, followed by Alexafluor 800-conjugated goat-antirabbit secondary antibody (green). Lanes of the blot correspond to (1) FP-biotin-treated NHP brain supernatant fraction #1 (200 μg), (2) FP-biotin treated NHP brain supernatant fraction #2 (9 μg), (3) FP-biotin-treated CES1 supersomes (3 μg), (4) NHP brain supernatant fraction #1 (200 μg), (5) NHP brain supernatant fraction #2 (9 μg), (6) CES1 supersomes (3 μg), (7) blank, and (8) molecular weight markers (size indicated on the right). The blot was simultaneously imaged with both 700 and 800 nm lasers, allowing simultaneous visualization of both fluorescent tags.

Article Snippet: Second, the membranes were probed with streptavidin conjugated to Alexafluor 680 (Thermo Fisher, S21378; diluted 1:4000 in blocking buffer containing 1% Tween 20) for 1 h at room temperature, followed by multiple washes with PBS, 0.05% Tween 20.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, SDS-Gel, Electrophoresis, Staining, Molecular Weight

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Schematic of the isNS1 batch immunoaffinity purification protocol and the downstream analyses used in this article. Infected cell supernatant from Vero cells (either WT or T164S EDEN2) was harvested at 72 hpi, clarified, filtered, supplemented with a protease inhibitor cocktail and 0.05% sodium azide, and finally concentrated using a 100 kDa MWCO Vivaflow cassette attached to a peristaltic pump. isNS1wt or isNS1ts was then batch immunoaffinity purified using the 56.2 anti-NS1 antibody immobilized on the AminoLink resin. The resin was then loaded into a column, washed with at least 10 CV of PBS (pH 7.4), eluted with 0.1 M glycine (pH 2.7), and immediately neutralized with 1 M Tris-HCl (pH 9.0). The eluted protein was then dialyzed against PBS and concentrated using a 100 kDa MWCO Amicon ultracentrifugal unit and stored at –80°C before use. The protein purity was determined via Coomassie blue after separation on a reducing SDS-PAGE. The protein bands observed on the gel were then validated in a western blot against NS1 and ApoA1. Protein quality was also determined via NS1 western blot following separation on a Native-PAGE. Excised bands corresponding to 250 kDa on the Native gel, and 50 kDa and 25 kDa bands on the denatured gel as well as the elute in solution were also subjected to protein identification via liquid chromatography mass spectrometry (LC-MS). Purified isNS1wt and isNS1ts were separately complexed with Fab56.2 and Ab56.2 and analyzed through an analytical size exclusion chromatography to ensure formation of stable complexes for imaging via electron microscopy. Purified isNS1wt and isNS1ts were also crosslinked with disuccinimidyl sulfoxide (DSSO) to determine interaction sites between isNS1 and ApoA1 via LC-MS. ( b ) Enrichment of NS1 during the immunoaffinity purification process. isNS1wt purification is used as a representative for the isNS1 purification process. The proportion of NS1 over total protein was measured by taking a percentage of the total amount of NS1 measured (using NS1 ELISA) out of the total protein measured (using Bradford assay) in the crude supernatant before concentrating, crude supernatant after concentrating, immunoaffinity PBS wash and finally in the immunoaffinity elute after buffer exchange against PBS and further concentrating. There is an approximately 455-fold enrichment of NS1 from the concentrated crude supernatant to the immunoaffinity elute. ( c ) Representative negative stain electron micrograph of isNS1 image on a 120 kV FEI Tecnai T12 equipped with an Eagle 4 mega pixel CCD camera. The corresponding 2D classes from the particles picked are shown on the right, red arrows highlighting the NS1 dimer protruding out of the spherical density.

Article Snippet: Vero cells (green African monkey kidney epithelial cells, ATCC) were cultured in DMEM containing 4.5 g/L glucose (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S) at 37°C in 5% CO 2 .

Techniques: Immunoaffinity Purification, Infection, Protease Inhibitor, Purification, SDS Page, Western Blot, Clear Native PAGE, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Size-exclusion Chromatography, Imaging, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Bradford Assay, Buffer Exchange, Staining

Journal: Autophagy

Article Title: The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity.

doi: 10.1080/15548627.2023.2247747

Figure Lengend Snippet: Figure 6. CLDN1 and MUC1 were degraded by lysosome in uterine lumenal epithelia during the establishment of uterine receptivity. (A) lysosomal fractionation and proteomics workflow. After uterine lumenal epithelial cells collection, lysosomes extraction, and trypsin digestion, peptides were identified by LC-MS/MS. (B) Schematic representation of quantitative proteomics for whole lumenal epithelial layer. (C) Enrichment analysis of the identified lysosomal proteins by Metascape (https://metascape.Org/). (D, E) Venn diagrams showing the overlap between protein with increase on D4 10:00 vs. 17:00 of uterine Le, proteins with reduction on D4 10:00 vs. 17:00 of uterine Le and proteins of lysosomes on D4 17:00 of uterine Le. (F) Heatmap of differentially expressed proteins (DEP) in uterine lumenal epithelia on D4 10:00/17:00. (G) Immunofluorescence analysis of CLDN1 (Green) and LAMP1 (red) in D4 uteri on 10:00 and 17:00. Scale bar: 20 μm. (H) Western blotting analysis of CLDN1 protein in uterine Le on D4 10:00/17:00. ACTB was used as a loading control. (I) Immunofluorescence analysis of CLDN1 (Green) and LAMP1 (red) in

Article Snippet: For immunofluorescence staining, 4% formaldehyde fixed frozen tissue section (10 μm) or 4% formaldehyde fixed tissue was processed for paraffin embedding, sectioned in 5-μm thickness was incubated with antibodies to LAMP1 (1:200; Abcam, Ab25245), CLDN1 (1:200; Santa Cruz Biotechnology, sc -81,796), or MUC1 (1:200; Abcam, Ab15481).

Techniques: Fractionation, Extraction, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Immunofluorescence, Western Blot, Control

Journal: Autophagy

Article Title: The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity.

doi: 10.1080/15548627.2023.2247747

Figure Lengend Snippet: Figure 7. Schematic roles of blastocyst derived signal facilitating lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity. In the presence of blastocyst, blastocyst-derived signal attended into the activation of epithelial p-STAT3, accompanied with upregulation of epithelial Lifr expression, to induce lysosomal hydrolase expression. The activated lysosomes in epithelia degraded CLDN1 and MUC1 proteins to facilitate the epithelial differentiation for the successful embryo implantation.

Article Snippet: For immunofluorescence staining, 4% formaldehyde fixed frozen tissue section (10 μm) or 4% formaldehyde fixed tissue was processed for paraffin embedding, sectioned in 5-μm thickness was incubated with antibodies to LAMP1 (1:200; Abcam, Ab25245), CLDN1 (1:200; Santa Cruz Biotechnology, sc -81,796), or MUC1 (1:200; Abcam, Ab15481).

Techniques: Derivative Assay, Transformation Assay, Activity Assay, Activation Assay, Expressing

Journal: Cell reports

Article Title: Piezo1 regulates colon stem cells to maintain epithelial homeostasis through SCD1-Wnt-β-catenin and programming fatty acid metabolism.

doi: 10.1016/j.celrep.2025.115400

Figure Lengend Snippet: Figure 1. Piezo1 expression and activity influence colon stem cell growth (A) Representative images of mouse colonoids treated with 10 mM GsMTX4, 2.5 mM Yoda1, or shPiezo1. Colonoids stably transfected with shRNA targeting the control and Piezo1 genes were defined as the shCtrl and shPiezo1 colonoids, respectively. Scale bar, 50 mm. (B and C) Statistical analysis of colon stem cell growth: the average surface area per colonoid (B) and the average number of buds per colonoid (C). (D and E) Immunofluorescence (IF) staining of EdU (D) and Ki67 (E) in colonoids. Scale bar, 50 mm. (F and G) Relative EdU and Ki67 signaling intensity in (D) and (E). (H) Western blot analysis of Piezo1 protein in colonoids transfected with shCtrl and shPiezo1. (A)–(H) were generated in colonoids from four mice, and at least three independent experiments were conducted. Results are described as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed unpaired Student’s t test). See also Figure S1.

Article Snippet: Colonoids were treated with DMSO (MCE), GsMTX4 (TargetMol, compared with PBS), Yoda1 (MCE, compared with DMSO), CAY10566 (TargetMol, compared with DMSO), and OA (TargetMol, compared with DMSO) in experiments.

Techniques: Expressing, Activity Assay, Stable Transfection, Transfection, shRNA, Control, Staining, Western Blot, Generated, Two Tailed Test

Journal: Cell reports

Article Title: Piezo1 regulates colon stem cells to maintain epithelial homeostasis through SCD1-Wnt-β-catenin and programming fatty acid metabolism.

doi: 10.1016/j.celrep.2025.115400

Figure Lengend Snippet: Figure 3. Piezo1 regulates colon stem cell stemness by mediating Wnt-b-catenin signaling through SCD1 (A) Volcano plot of differentially expressed genes (DEGs) between PBS (control) and GsMTX4-treated (determined via RNA-seq) colonoids. The genes were selected via | log2 (fold change) | > 1 and p < 0.05. (B) Gene Ontology (GO) analysis of DEGs, including biological process (BP), cell component (CC), and molecular function (MF). (C) Western blot of SCD1 protein in colonoids treated with Yoda1 or GsMTX4. (D) Representative images of colonoids treated with CAY10566 (5 nM), GsMTX4, or GsMTX4 and CAY10566. The bottom row shows the Lgr5-EGFP staining. Scale bar, 100 mm. (E) Experimental design for different interventions in (D).

Article Snippet: Colonoids were treated with DMSO (MCE), GsMTX4 (TargetMol, compared with PBS), Yoda1 (MCE, compared with DMSO), CAY10566 (TargetMol, compared with DMSO), and OA (TargetMol, compared with DMSO) in experiments.

Techniques: Control, RNA Sequencing, Western Blot, Staining

Journal: Cell reports

Article Title: Piezo1 regulates colon stem cells to maintain epithelial homeostasis through SCD1-Wnt-β-catenin and programming fatty acid metabolism.

doi: 10.1016/j.celrep.2025.115400

Figure Lengend Snippet: Figure 5. Exogenous OA restored impaired colon stem cell stemness in Yoda1-treated mice (A) IHC staining of Ki67 in WT and Yoda1 mouse colons. Statistical analysis of the Ki67+ cell ratio is shown on the right. Scale bar, 20 mm. (B) IF co-staining of Lgr5-EGFP and Ki67 in mouse colons. Statistical analysis of the Lgr5+ cell number and the proliferating Lgr5+ cell ratio is shown on the right. Scale bar, 20 mm. (C) Western blot of Lgr5, b-catenin, and Axin2 proteins in mouse colon crypts. (D) Representative images of colonoids from WT and Yoda1 mice. Colonoids from Yoda1 mice were treated with 50 mM OA for 72 h, and OA was added into the medium immediately after seeding. Scale bar, 100 mm. (E) IF staining of Lgr5-EGFP in colonoids. Scale bar, 100 mm. (F) Statistical analysis of the average surface area per colonoid, the average number of buds per colonoid, and the GFP+ cell ratio in colonoids. (G) IF co-staining of EdU and Ki67 in colonoids. Scale bar, 100 mm. (H) RNA expression of Lgr5, b-catenin, and Axin2 in colonoids. (A) and (B) were generated from five mice, (C) was generated from three mice, and (D)–(H) were generated in colonoids from three mice per group. The results are representative of at least three independent experiments. Results are described as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed unpaired Student’s t test or multiple comparisons with ANOVA test). See also Figure S6.

Article Snippet: Colonoids were treated with DMSO (MCE), GsMTX4 (TargetMol, compared with PBS), Yoda1 (MCE, compared with DMSO), CAY10566 (TargetMol, compared with DMSO), and OA (TargetMol, compared with DMSO) in experiments.

Techniques: Immunohistochemistry, Staining, Western Blot, RNA Expression, Generated, Two Tailed Test

Journal: Cell reports

Article Title: Piezo1 regulates colon stem cells to maintain epithelial homeostasis through SCD1-Wnt-β-catenin and programming fatty acid metabolism.

doi: 10.1016/j.celrep.2025.115400

Figure Lengend Snippet: Figure 6. Knockdown or activation of Piezo1 reprogrammed fatty acid metabolism in mouse colon crypts (A) Western blot of SCD1 protein in WT, Piezo1DISC, and Yoda1 mouse colon crypts. (B) Heatmap of the FA content in WT, Piezo1DISC, and Yoda1 mouse colon crypts through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Compared with WT mice, the increased FAs in Piezo1DISC mice are marked in red, and the decreased FAs in Yoda1 mice are marked in blue. The FAs that were increased in Piezo1DISC mice while decreased in Yoda1 mice are marked in orange. (C) Percentages of SFA, MUFA, PUFA, and TFA contents in different groups. (D) The ratio of overall MUFAs and PUFAs to SFAs in different groups. The ratio reflects the overall desaturation of MUFAs and PUFAs. (E) The ratio of POA, PSA, and OA contents in MUFAs and the ratio of ALA, GLA, and AA in PUFAs in different groups. (F) The desaturation index of POA (C16:1/C16:0), PSA (C18:1(n–12)/C18:0), OA (C18:1(n–9)/C18:0), and AA (C20:4/C20:0) in different groups. (A)–(F) were generated from three mice per group. Results are described as the mean ± SEM. ns, not significant, *p < 0.05, and **p < 0.01 (multiple comparisons with ANOVA test).

Article Snippet: Colonoids were treated with DMSO (MCE), GsMTX4 (TargetMol, compared with PBS), Yoda1 (MCE, compared with DMSO), CAY10566 (TargetMol, compared with DMSO), and OA (TargetMol, compared with DMSO) in experiments.

Techniques: Knockdown, Activation Assay, Western Blot, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Applied and Environmental Microbiology

Article Title: Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation

doi: 10.1128/AEM.02068-17

Figure Lengend Snippet: Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining. The stained biofilm was released using 95% ethanol and determined by monitoring the value of OD595. (B to D) The concentration of eDNA during biofilm formation under optimal conditions (B), aerobic conditions (C), and starvation conditions (D) over 3 days was quantified using SYBR green I dye on the basis of a standard curve generated using a series of 10-fold dilutions of Lambda DNA from 80 μg/ml to 0.156 μg/ml.

Article Snippet: C. jejuni F38011 was used as the representative strain for the following study due to its intense biofilm formation and remarkable response to different environmental conditions. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 1 caption a7 Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining.

Techniques: Staining, Concentration Assay, SYBR Green Assay, Generated, Lambda DNA Preparation

Journal: Applied and Environmental Microbiology

Article Title: Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation

doi: 10.1128/AEM.02068-17

Figure Lengend Snippet: Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni F38011 and the corresponding spoT, recA, and flaAB deletion mutants under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining. The stained biofilm was released using 95% ethanol and determined by monitoring the value of OD595. (B to D) The concentration of eDNA during biofilm formation under optimal conditions (B), aerobic conditions (C), and starvation conditions (D) over 3 days was quantified using SYBR green I dye on the basis of a standard curve generated using a series of 10-fold dilutions of Lambda DNA from 80 μg/ml to 0.156 μg/ml.

Article Snippet: C. jejuni F38011 was used as the representative strain for the following study due to its intense biofilm formation and remarkable response to different environmental conditions. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 1 caption a7 Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining.

Techniques: Staining, Concentration Assay, SYBR Green Assay, Generated, Lambda DNA Preparation

Journal: Applied and Environmental Microbiology

Article Title: Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation

doi: 10.1128/AEM.02068-17

Figure Lengend Snippet: Confocal micro-Raman spectroscopy monitors the development of C. jejuni F38011 biofilm in the microfluidic “lab-on-a-chip” platform. C. jejuni F38011 biofilm was cultivated in a microfluidic device, and the chemical component was determined at 72 h, 120 h, 168 h, and 216 h using confocal micro-Raman spectroscopy coupled with a 532-nm-wavelength laser. (A) Prominent Raman peaks during biofilm formation. (B) Variations in the intensity of the corresponding Raman peaks (746, 918, 968, 1,123, 1,168, 1,370, 1,580, and 1,643 cm−1) over time. The Raman peaks derived from nucleic acid components (746 and 1,580 cm−1) are shown in black, and the Raman peaks derived from other components (i.e., proteins, lipids, and polysaccharides) are shown in gray.

Article Snippet: C. jejuni F38011 was used as the representative strain for the following study due to its intense biofilm formation and remarkable response to different environmental conditions. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 1 caption a7 Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining.

Techniques: Raman Spectroscopy, Lab-on-a-Chip, Derivative Assay

Journal: Applied and Environmental Microbiology

Article Title: Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation

doi: 10.1128/AEM.02068-17

Figure Lengend Snippet: Addition of genomic DNA extracted either from Campylobacter or Salmonella had a concentration-dependent stimulation effect on biofilm formation of C. jejuni F38011; the precoating layer formed by DNA extracted either from Campylobacter or Salmonella did not contribute to the development of biofilm formation of C. jejuni F38011. Genomic DNA of C. jejuni F38011 or S. Typhimurium SL1344 was separately extracted and added for biofilm formation. (A and B) To form a precoating layer, 200 μl of DNA of Salmonella (A) or C. jejuni (B) at different concentrations was added into each well of the 96-well plate and maintained for 4 h. The unbounded DNA was washed out before the addition of C. jejuni F38011 culture. (C and D) To directly add DNA for biofilm formation, DNA of Salmonella (C) or C. jejuni (D) was mixed with C. jejuni F38011 culture to reach a certain final concentration and 200 μl of this mixed culture was added into the 96-well plate. The plate was then cultivated in a microaerobic environment at 37°C for up to 72 h.

Article Snippet: C. jejuni F38011 was used as the representative strain for the following study due to its intense biofilm formation and remarkable response to different environmental conditions. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 1 caption a7 Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining.

Techniques: Concentration Assay

Journal: Applied and Environmental Microbiology

Article Title: Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation

doi: 10.1128/AEM.02068-17

Figure Lengend Snippet: DNase I treatment reduced the coverage and volume of C. jejuni biofilm. The well-developed (3-day) C. jejuni F38011 biofilm on a nitrocellulose membrane was treated with DNase I solution (2 U/ml) for 15 min and then air-dried for the analysis of atomic force microscopy. (A) Coverage reduction caused by DNase I treatment. (B) Volume reduction caused by DNase I treatment. Asterisks denote significant difference (P < 0.05).

Article Snippet: C. jejuni F38011 was used as the representative strain for the following study due to its intense biofilm formation and remarkable response to different environmental conditions. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 1 caption a7 Biofilm formation and release of extracellular DNA (eDNA) by wild-type C. jejuni strains (i.e., human 10, 81-116, ATCC 33560, 87-95, NCTC 11168, 1658, and F38011) under optimal, aerobic, and starvation conditions. (A) The level of biofilm formation was evaluated using crystal violet staining.

Techniques: Microscopy

Journal: Nature Communications

Article Title: Cell facilitation promotes growth and survival under drug pressure in breast cancer

doi: 10.1038/s41467-023-39242-6

Figure Lengend Snippet: a Growth of CAMA-1 spheroids under drug pressure with supplemented conditioned media from spheroids of different compositions. Sensitive and resistant cells were plated by the indicated composition of sensitive and resistant cells in a 3D spheroid format and underwent exposure to untreated or 400 nM treated medium to produce conditioned media. 100% sensitive spheroids under drug pressure were then supplemented with different concentrations of conditioned media. Normalized cell counts of sensitive cells supplemented with untreated or treated conditioned media generated from different spheroid compositions. b Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for estradiol detection performed on CAMA-1 untreated or ribociclib 200 nM treated samples of the following compositions at Day 21: 100% sensitive, 50% sensitive–50% resistant, 100% resistant samples. Box plots are plotted as mean with SD, the boxes range from the 25th to 75th percentile, the whiskers range from the min to max value. Sample size is n = 3 for each case. Comparison of estradiol concentration differences between untreated and treated conditions in 50%–50% coculture media ( p = 0.0195 with a two-tailed t -test (t = 7.061). c Michaelis–Menten model of estradiol production and uptake fitted to estradiol concentrations in sensitive (S cells: stars) or resistant (R cells: open circles) monocultures and cocultures (Mixed: filled circles) when treated with (red) or without (black) ribociclib. Large points indicate means within treatment groups, small points = replicate measurements. Comparing observed versus predicted estradiol concentrations (left panel) shows the model captured differential estradiol fluxes of sensitive and resistant monocultures and cocultures. Variance explained = 85%, fit line slope = 0.999, adjusted \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}^{2}$$\end{document} R 2 = 0.85). Comparing estimated estradiol production and uptake of resistant and sensitive cells (right panel points) indicates that resistant cells produce ~8 times more estradiol and use only 2.5 times more than sensitive. Parameter uncertainty is indicated by 95% confidence intervals (CI = error bars). d Diagram depicting CAMA-1 resistant and sensitive cell contributions of estradiol metabolism enzymes Aromatase, HSD17β1, and HSD17β8. e Western blot analysis of Aromatase, HSD17β1, HSD17β8, phosphorylated-ER, total ER, and β-actin (control) in monoculture, untreated sensitive and resistant CAMA-1 cells. Western Blots were performed in triplicates with consistent findings. Source data provided in Source Data file.

Article Snippet: After washing with TTBS, membranes were then probed with anti-aromatase (Invitrogen, MA5-32628, 1:7000 dilution, overnight 4 °C), anti-HSD17β1 polyclonal antibody (Abnova, H00003292-M03A, 1:1000 dilution, overnight 4 °C; R&D systems, MAB7178, 1:2000 dilution, overnight 4 °C), anti-HSD17β8 polyclonal antibody (Proteintech, 16752-1-AP, 1:1000 dilution, overnight 4 °C), anti-ER (Cell Signaling, 8644S, 1:3000 dilution, overnight 4 °C), anti-phospho-ER (Cell Signaling, 2511S, 1:500 dilution, overnight 4 °C) and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, sc-47778, 1:500 dilution, 1 h room temperature) and detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) with anti-rabbit (GE Healthcare NA9341ML) or anti-mouse (GE Healthcare NXA9311ML) peroxidase-linked secondary antibody (1:6000 dilution).

Techniques: Generated, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Comparison, Concentration Assay, Two Tailed Test, Western Blot, Control

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: HDAC 3-selective inhibitor RGFP966 attenuates LPS/IFNγ-induced NF-κB p65 activity but affects neither the NF-κB p65 acetylation nor the histone acetylation status in RAW 264.7 macrophages. RAW-Blue cells were subjected to RGFP966 and SAHA for 20 h and stimulated with LPS/IFNγ for the last 4 h of the experiment. In the absence of an inflammatory stimulus, treatment of RAW-Blue cells with HDAC inhibitors did not affect the secretion of embryonic alkaline phosphatase (SEAP; data not shown), whereas treatment of RAW-Blue cells with RGFP966 followed by a pro-inflammatory stimulus significantly reduced the secretion of SEAP (A). Data are presented as mean values ± SD of 3–4 independent experiments. *** p < 0.001, compared to vehicle-treated group. The effect of HDAC 3 inhibition on total NF-κB p65 acetylation was assessed by (B) immunoblotting and quantified by densitometric analysis (C). RAW 264.7 macrophages were incubated with HDAC inhibitors for 20 h and subsequently lysed. For the detection of NF-κB p65 acetylation, lysates were incubated and immunoprecipitated with 5 μg of mouse monoclonal NF-κB p65-specific antibody as described. Data are presented as mean values ± SD expressed as fold change compared to control (vehicle-treated) group of 4 independent experiments. Histones were separated on SDS–PAGE (D), subsequently histones H3 and H4 were excised from the gel. Gel pieces were treated with acetic anhydride d6, followed by trypsin digestion. Resulting peptides were subjected to LC–MS/MS analysis (E). RGFP966 treatment did not affect the acetylation status of histone H3 (res. peptide 18–26: KQLATKAAR) and histone H4 (res. peptide 4–17: GKGGKGLGKGGAKR), whereas SAHA increased acetylation status of both peptides. Data are presented as mean values ± SD of 3–5 independent experiments. No significant differences were observed between untreated and vehicle treated cells (data not shown).

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Activity Assay, Inhibition, Western Blot, Incubation, Immunoprecipitation, Control, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: RGFP966 does not affect the nuclear translocation of NF-kB p65 in RAW 264.7 macrophages. After 20 h incubation with RGFP966 followed by 1 h LPS/IFNγ stimulation, RAW 264.7 macrophages were prepared for immunofluorescence microscopy (A). Green signal represents NF-kB p65 protein, while the blue signal visualizes the Hoechst-stained nuclei. RGFP966 did not affect the nuclear translocation of NF-kB p65 compared to (vehicle-treated) control group. The presented data set shows representative images of 3 independent experiments, original magnification 400×. All images were taken under identical instrumental conditions. In addition, the effect of RGFP966 on NF-kB p65 translocation in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting cell fractions (B) and quantified by densitometric analysis (C). PARP-1 and β-actin were used as internal controls. Data are presented as mean values ± SD expressed as fold change compared to control (LPS/IFNγ-treated) group of 4 independent experiments. RAW 264.7 macrophages were stimulated with 10 ng/ml LPS/IFNγ for 15, 30 and 60 min and subsequently lysed. In parallel, to confirm proper LPS/IFNγ-stimulation and to determine the levels of IκBα in the presence of RGFP966, IκBα expression was assessed by Western blotting (D). β-Actin was used as loading control. The presented data set shows a representative blot of 3 independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Translocation Assay, Incubation, Immunofluorescence, Microscopy, Staining, Control, Western Blot, Expressing

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: HDAC 3-selective inhibitor RGFP966 reduces the protein expression of HDAC 1 and HDAC 2 in RAW 264.7 macrophages. RAW 264.7 macrophages were incubated with RGFP966, stimulated for 4 h with LPS/IFNγ and subsequently harvested. Gene expression of NF-kB p65, IκBα, HDAC 1, HDAC 2 and HDAC 3 was analyzed by real-time qPCR (A). Data are presented as mean values ± SD; n = 3–4. In parallel, the effect of RGFP966 on total NF-kB p65 and HDAC 1–3 protein expression in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting (B) and quantified by densitometric analysis (C). Protein levels were normalized against β-actin and control (LPS/IFNγ-treated) cells were set at 100%. Data are presented as mean values ± SD of 4 independent experiments and a representative blot is shown in B. * p < 0.05; ** p < 0.01 compared to vehicle.

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Expressing, Incubation, Gene Expression, Western Blot, Control

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: RGFP966 does not affect the localization of HDAC 3 and HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RAW 264.7 macrophages. After 20 h incubation with RGFP966 followed by 1 h LPS/IFNγ stimulation, RAW 264.7 macrophages were prepared for immunofluorescence microscopy (A). The green signal represents NF-kB p65 protein, while the blue signal visualizes the Hoechst-stained nuclei. RGFP966 did not affect the nuclear localization of HDACs 1–3 compared to (vehicle-treated) control group. The presented data set shows representative images of 4 independent experiments. Images of the cells were taken using confocal laser scanning microscopy and all images were taken with identical instrumental conditions, original magnification 630×. In addition, the effect of RGFP966 on HDAC 1–3 localization in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting cell fractions (data not shown) and quantified by densitometric analysis (B). PARP-1 and β-actin were used as internal controls. Control (vehicle-treated) cells were set at 100%. Data are presented as mean values ± SD of 3–4 independent experiments. The HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RGFP966-treated RAW 264.7 macrophages was investigated by immunoprecipitation of NF-κB p65 followed by immunoblotting for HDAC 3, which was quantified by densitometric analysis (C). Protein levels were normalized against NF-κB p65. Data are presented as mean values ± SD of 3 independent experiments and a representative blot is shown. * p < 0.05 compared to vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Incubation, Immunofluorescence, Microscopy, Staining, Control, Confocal Laser Scanning Microscopy, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: Tolterodine is a novel candidate for assessing CYP3A4 activity through metabolic volatiles to predict drug responses

doi: 10.1038/s41598-025-86450-9

Figure Lengend Snippet: ( a ) A chromatogram illustrating the production of two major metabolites 5-hydroxymethyl tolterodine (predominantly from the CYP2D6 isoform) and N-dealkylated tolterodine (predominantly from CYP2C9 and CYP3A4). ( b ) Biotransformation of tolterodine after 4 h incubation time with three different CYP overexpressing HepG2 cell clones and the corresponding empty vector (EV) cell clones to N-dealkylated tolterodine measured by LC-MS. *** p < 0.001 in comparison to the HepG2-CYP3A4 cells.

Article Snippet: The metabolite 5-hydroxymethyl tolterodine (99.05%) was supplied by MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Incubation, Clone Assay, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Comparison