Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: Gut microbiota induces sphingolipid pathway disturbance in the peripheral and brain. ( A ) Representative metabolites classified and counted based on their chemical taxonomy in shScr and shCry2 mice. The size of the pie chart corresponded to the relative abundance levels of metabolites, (n = 10 mice/group)., (B) Volcano plot of differentially expressed metabolites from shScr versus shCry2 mice. Blue, red, and grey represented downregulated, upregulated, and no significantly expressed metabolites, respectively, (n = 10 mice/group)., ( C ) Unsupervised hierarchical clustering of differentially expressed metabolites from shScr and shCry2 mice, (n = 10 mice/group)., ( D ) Pathway enrichment analysis of differentially expressed metabolites between shScr and shCry2 mice, (n = 10 mice/group)., ( E ) Representative Western blot and quantification of Sphk1 and S1PR1 in the hippocampus of shScr and shCry2 mice., ( F ) LC/MS analysis of Cer(18:1/18:0) and Cer(18:2/18:0) in shScr and shCry2 mice., ( G ) Linear regression analyses between sphingosine and Akkermansia, (left plot) and Ruminococcaceae, (right plot) expression levels from data integrated from 16 S rRNA sequencing and metabolomic sequencing. All data were expressed as mean ± SEM. Data were considered significant if * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Means were compared using the Student’s t-test in panel, ( E , F ). shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing, Sequencing, Metabolomic, IF-P

Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: FTY720 ameliorated the cognitive decline, impairment of BBB and tau pathology. ( A ) An overview of the experimental design. After receiving stereotaxic brain injections, three-month-old mice were housed for six weeks, followed by intraperitoneal injections of saline or FTY720 for two weeks. Behavioral and molecular biology tests were then conducted., ( B - E ) Escape latency of the shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice for 5 consecutive days in the MWM test, ( B ). Percentage of time spent in the target quadrant in the spatial probe trail of the MWM test, ( C ). Number of platform crossings in the spatial probe trail of the MWM test, ( D ). Representative heatmaps of the swimming path in the spatial probe trail of day 6 in the MWM test, ( E ), (n = 11–12 mice/group)., ( F - H ) Representative Western blot and quantification of Occludin, ZO-1, ( F ), Sphk1, S1PR1, ( G ), Tau1, pS396-tau, Tau5, and pT231-tau, ( H ) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice., ( I , J ) Representative immunofluorescence staining and quantification of pS396-tau, (red) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice. Magnification × 10. Scale bar = 100 μm. All data were expressed as mean ± SEM. Data were considered significant if *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Means were compared using a repeated measures 2-way ANOVA test with Bonferroni post hoc comparison in panel, ( B ) and one-way ANOVA, ( C - D , F - I ). FI: Fluorescence intensity. shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Saline, Western Blot, Immunofluorescence, Staining, IF-P, Comparison, Fluorescence

Journal: Cell

Article Title: ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P

doi: 10.1016/j.cell.2018.08.020

Figure Lengend Snippet: A) Indicated expression vectors were transfected into HEK293 cells. After 48 hr, cells were incubated with 25 μg/ml N-acetyl-leucine-leucine-norleucinal (ALLN) for 3 hr before harvest. WCL were IB’d with antibodies to SREBP1 and 2. B) Indicated expression vectors were transfected into SCAP-ablated HEK293 cells (293ΔSCAP). After 5 hr, cells were incubated with 1% lipoprotein-deficient serum (LPDS) for 16 hr, followed by ethanol or 50 μM mevalonate + 5 μg/ml cholesterol for 12 hr and 3 hr with ALLN before harvest. WCL, membranes, and NE were IB’d. Red stars: N-terminal Myc epitope-containing S1P polypeptides. C) Intracellular localization of Casp2-HA, S1P-Myc and Flag-SREBP2 in S1P-ablated HEK293 cells (293ΔS1P). Transiently expressed Casp2, S1P, and SREBP2 were visualized by immunofluorescence with HA, Myc, and FLAG antibodies, respectively. Nuclei were counterstained with DAPI. Scale bar: 10 μm. D) Indicated proteins were expressed in HEK293 cells. After 48 hr, WCL and culture supernatants (CS) were IB’d with Myc and S1P antibodies (2 lanes per condition). E) Indicated proteins were expressed in 293ΔSCAP cells. 5 hr after transfection, the cells were incubated with 1% LPDS for 16 hr followed by 3 hr with ALLN. WCL, membranes, and NE were prepared and Casp2 and S1P were IB’d with HA and Myc antibodies. CS were IB’d for S1P. Arrow: cleaved Casp2, red stars: N-terminal S1P fragments that retained the Myc epitope. F) ER and Golgi fractions were isolated by differential centrifugation from livers of LFD-fed 7-week-old MUP-uPA and Casp2−/−/MUP-uPA mice. Proteins in each fraction were de-glycosylated with PNGase F and IB’d as indicated. Star: nonspecific band present in the Golgi fraction of Casp2-null liver, F.L: full-length, C2-Cl.: Casp2-cleaved, A-Cl.: autocleaved. G) BL6 hepatocytes introduced with Adv carrying GFP or uPA. After 24 hr, ER, Golgi and nuclear fractions were isolated by differential centrifugation and IB’d for indicated proteins. F.L: full-length, Cl.: cleaved, L.E: long-exposure, S.E: short-exposure. H) IB of S1P in sera of HFD-fed (12 weeks) mice (n = 3) of indicated genotypes. I) Sera from normal individuals, NAFLD and NASH patients (n = 3 per group) with different liver fibrosis scores were IB’d with S1P antibody.

Article Snippet: Mouse S1P (MC204593) and mouse Casp2 cDNA clone (MC206011) were purchased from Origene Technologies, Inc. Flag-tagged SREBP2 (#32018) cDNA clone was from Addgene.

Techniques: Expressing, Transfection, Incubation, Immunofluorescence, Isolation, Centrifugation

Journal: Drug Design, Development and Therapy

Article Title: Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence

doi: 10.2147/DDDT.S343149

Figure Lengend Snippet: The Primer Sequences of Relative Genes

Article Snippet: Primary antibodies used were as follows: S1P (Bioss, Beijing, China); S1PR5 (Proteintech, Wuhan, China); GNAI1 (Proteintech, Wuhan, China); Rac1 (Proteintech, Wuhan, China); KRAS (Proteintech, Wuhan, China); ERK1/2 (Proteintech, Wuhan, China); RAF1 (Proteintech, Wuhan, China); Phospho-ERK1/2 (Proteintech, Wuhan, China); MEK1 (Santa, USA); Phospho-Raf1 (Abcam, USA); Phospho-MEK1 (Abcam USA); PI3K p85 alpha (Proteintech, Wuhan, China), Phospho-PI3K p85 alpha (Abcam USA); RAC1 (Proteintech, Wuhan, China); GAPDH (Proteintech, Wuhan, China).

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence

doi: 10.2147/DDDT.S343149

Figure Lengend Snippet: YHT suppressed Lewis lung carcinoma recurrence by inhibiting the sphingolipid signaling pathway. ( A and B ) qRT-PCR analysis of S1P, S1PR5, GNAI1, PI3K, RAC1, KRAS, RFA1, MEK1 and ERK1 expression in Lewis lung carcinoma mice after the indicated 14 days treatments; ( C – F ) Western blotting analysis of S1P, S1PR5, GNAI1, PI3K, RAC1, KRAS, RFA1, MEK1 and ERK1/2 expression in Lewis lung carcinoma mice after the indicated 14 days treatments. Error bars are means ± SD, n= 3 randomly selected magnification fields. P values were calculated using two-tailed unpaired Student’s t -test. * P <0.05 and ** P <0.01.

Article Snippet: Primary antibodies used were as follows: S1P (Bioss, Beijing, China); S1PR5 (Proteintech, Wuhan, China); GNAI1 (Proteintech, Wuhan, China); Rac1 (Proteintech, Wuhan, China); KRAS (Proteintech, Wuhan, China); ERK1/2 (Proteintech, Wuhan, China); RAF1 (Proteintech, Wuhan, China); Phospho-ERK1/2 (Proteintech, Wuhan, China); MEK1 (Santa, USA); Phospho-Raf1 (Abcam, USA); Phospho-MEK1 (Abcam USA); PI3K p85 alpha (Proteintech, Wuhan, China), Phospho-PI3K p85 alpha (Abcam USA); RAC1 (Proteintech, Wuhan, China); GAPDH (Proteintech, Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Journal: Drug Design, Development and Therapy

Article Title: Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence

doi: 10.2147/DDDT.S343149

Figure Lengend Snippet: Binding Free Energies and Energy Components Predicted by MM/GBSA (kcal/mol)

Article Snippet: Primary antibodies used were as follows: S1P (Bioss, Beijing, China); S1PR5 (Proteintech, Wuhan, China); GNAI1 (Proteintech, Wuhan, China); Rac1 (Proteintech, Wuhan, China); KRAS (Proteintech, Wuhan, China); ERK1/2 (Proteintech, Wuhan, China); RAF1 (Proteintech, Wuhan, China); Phospho-ERK1/2 (Proteintech, Wuhan, China); MEK1 (Santa, USA); Phospho-Raf1 (Abcam, USA); Phospho-MEK1 (Abcam USA); PI3K p85 alpha (Proteintech, Wuhan, China), Phospho-PI3K p85 alpha (Abcam USA); RAC1 (Proteintech, Wuhan, China); GAPDH (Proteintech, Wuhan, China).

Techniques: Binding Assay

Journal: Drug Design, Development and Therapy

Article Title: Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence

doi: 10.2147/DDDT.S343149

Figure Lengend Snippet: Molecular docking of three pivotal active ingredients and S1PR5. ( A ) S1PR5 and stigmasterol; ( B ) S1PR5 and nootkatone; ( C ) S1PR5 and ergotamine.

Article Snippet: Primary antibodies used were as follows: S1P (Bioss, Beijing, China); S1PR5 (Proteintech, Wuhan, China); GNAI1 (Proteintech, Wuhan, China); Rac1 (Proteintech, Wuhan, China); KRAS (Proteintech, Wuhan, China); ERK1/2 (Proteintech, Wuhan, China); RAF1 (Proteintech, Wuhan, China); Phospho-ERK1/2 (Proteintech, Wuhan, China); MEK1 (Santa, USA); Phospho-Raf1 (Abcam, USA); Phospho-MEK1 (Abcam USA); PI3K p85 alpha (Proteintech, Wuhan, China), Phospho-PI3K p85 alpha (Abcam USA); RAC1 (Proteintech, Wuhan, China); GAPDH (Proteintech, Wuhan, China).

Techniques:

Journal: Reproductive Medicine and Biology

Article Title: Sphingosine kinases negatively regulate the expression of matrix metalloproteases ( MMP1 and MMP3 ) and their inhibitor TIMP3 genes via sphingosine 1‐phosphate in extravillous trophoblasts

doi: 10.1002/rmb2.12379

Figure Lengend Snippet: Extracellular S1P transport in the cytoplasm of HTR‐8/SVneo cells. A, Cells were treated with TAMRA‐S1P (1 µM, red, left panel) for 1 h, washed with PBS, and then nuclei were stained with Hoechst 33258 (blue, middle panel). The overlay is shown in the right panel. TAMRA‐S1P was able to enter the cells within 30 minutes

Article Snippet: TAMRA‐S1P was from Echelon Biosciences.

Techniques: Staining