Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Demographics and characteristics of patients with oral squamous cell carcinoma.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Staining

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Overexpression of SPNS2 protein in primary OSCC tissues as detected by immunohistochemical staining. Representative immunohistochemical staining of SPNS2 protein indicating negative or positive cytoplasmic expression: ( left ) Normal tissue from adjacent to the tumor, and ( right ) Primary tumor tissue. Magnification 100× ( top ) and 400× ( bottom ). Scale bars: 25 μm ( top ) and 100 μm ( bottom ).

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Over Expression, Immunohistochemical staining, Staining, Expressing

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Clinicopathologic variables correlated with SPNS2 expression in patients with oral squamous cell carcinoma.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Kaplan–Meier survival analysis of negative and positive cytoplasmic SPNS2 protein expression in stage III/IV OSCC patients for use in log-rank tests of homogeneity using Kaplan–Meier curves. * p < 0.05.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Overall survival of III/IV stage and clinicopathologic variables of patients with oral squamous cell carcinoma using univariate and multivariate analysis.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Oncogene

Article Title: β3-adrenoreceptor blockade reduces tumor growth and increases neuronal differentiation in neuroblastoma via SK2/S1P 2 modulation

doi: 10.1038/s41388-019-0993-1

Figure Lengend Snippet: β3-AR blockade modulates S1P signaling in human neuroblastoma BE(2)C cells. a RT-PCR of human NB BE(2)C cell line treated with 1 μM SR59230A for 24 h. Change of mRNA expression levels of receptors (S1P 1 , S1P 2 , S1P 3 ), metabolic enzymes (SK1, SK2, SPL) and transporter (Spns2) of S1P are reported as mean ± SD of three independent experiments performed in triplicate, using the 2^(-ΔΔCt) method as described in Methods section. Data were normalized to β-actin RNA expression and values of treated samples reported as fold change over control, set as 1. Significance was calculated by Unpaired t -test analysis with equal SD (** P < 0.01). b WB and relative densitometric quantification analysis, showing protein expression levels of SK1, SK2, and S1P 2 in NB BE(2)C cell line after 24 h of 1 μM SR59230A treatment. Results were normalized to the expression of β-actin and reported as mean ± SD, fold change over control, set as 1. Blots are representative of three independent experiments. Significance was calculated by Unpaired t -test analysis with equal SD (* P < 0.05, *** P < 0.001). c WB and relative densitometric quantification analysis, showing protein expression levels of β2-AR, β3-AR, SK2 and S1P 2 in NB BE(2)C cell line after molecular silencing of β2- and β3-AR. Results were normalized to the expression of β-actin and reported as mean ± SD, fold change over control, set as 1. Blots are representative of three independent experiments. Significance was calculated by one-way ANOVA analysis followed by Bonferroni’s post hoc test (ns = not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001)

Article Snippet: Simultaneous amplification of the target sequences (SPHK1:Hs00184211_m1, SPHK2:Hs01016543_g1, SGPL1:Hs00393705_m1, SPNS2:Hs01390449_g1, S1PR1:Hs00173499_m1, S1PR2:Hs01003373_m1, S1PR3:Hs01019574_m1 Gene Expression assays) together with the housekeeping gene, β-actin (ACTB: Hs99999903_m1 Gene Expression assay) was carried out essentially as previously described [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Control

Journal: Nature communications

Article Title: Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2.

doi: 10.1038/s41467-025-55942-7

Figure Lengend Snippet: Fig. 2 | DDM binding pocket of SPNS2. a Bound n-dodecyl-β-D-maltopyranoside within the SPNS2 structure determined in DDM. Positive difference density of the weighted Fo −Fc difference map at 16σ and the DDM model are shown as blue surface and DDM cyan sticks, respectively. b Cross section of the SPNS2 structure, viewed from the plane of the membrane. c SPNS2 coordinates DDM through van der Waals contacts in the pocket and hydrogen bonds in the central cavity. SPNS2-

Article Snippet: Briefly, codon-optimized sequences of wild type and mutant SPNS2 in pDONR221 (Addgene #132307) were subcloned into a modified pJTI R4 DEST CMV TO pA plasmid (Thermo Fisher Scientific) containing Twin-Strep-Tag (IBA Lifesciences) and HA epitopes (SH tag) at the N or the C terminus of SPNS2 as indicated.

Techniques: Binding Assay, Membrane

Journal: Nature communications

Article Title: Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2.

doi: 10.1038/s41467-025-55942-7

Figure Lengend Snippet: Fig. 3 | Substrate and inhibitor binding by SPNS2. a Sphingosine-1-phosphate head group interactions with SPNS2 in simulation cluster 1. Carbons of SPNS2 side chains and FTY720-P are shown in purple and cyan, respectively. Interaction types are annotated by PLIP77. b FTY720-P interactions with SPNS2 in simulation cluster 1. c Inhibition of S1P export by 33p measured by S1PR3-coupled export assay after

Article Snippet: Briefly, codon-optimized sequences of wild type and mutant SPNS2 in pDONR221 (Addgene #132307) were subcloned into a modified pJTI R4 DEST CMV TO pA plasmid (Thermo Fisher Scientific) containing Twin-Strep-Tag (IBA Lifesciences) and HA epitopes (SH tag) at the N or the C terminus of SPNS2 as indicated.

Techniques: Binding Assay, Inhibition

Journal: Nature communications

Article Title: Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2.

doi: 10.1038/s41467-025-55942-7

Figure Lengend Snippet: Fig. 5 | Pathogenic mutations at essential locations in the SPNS2 structures. a Locations of pathogenic SPNS2 mutations within the structure. Loop 7-8 is not resolved in the structure, and the approximate location of Pro356 is indicated by a magenta sphere with a dotted edge. b The pathogenic mutation ΔS319 within the C-domain. c Asp163 in SPNS2 is within the conserved MFS motif A and forms hydrogen bonds specific to the outward-facing conformation (PDB: 8EX5). Hydrogen bonds are shown as dotted lines. d S1P export activity by pathogenic

Article Snippet: Briefly, codon-optimized sequences of wild type and mutant SPNS2 in pDONR221 (Addgene #132307) were subcloned into a modified pJTI R4 DEST CMV TO pA plasmid (Thermo Fisher Scientific) containing Twin-Strep-Tag (IBA Lifesciences) and HA epitopes (SH tag) at the N or the C terminus of SPNS2 as indicated.

Techniques: Mutagenesis, Activity Assay

Journal: Mucosal immunology

Article Title: Sphingosine-1-phosphate receptor-1 (S1P 1 ) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease

doi: 10.1038/mi.2016.35

Figure Lengend Snippet: Analyses of mRNA expression of enzymes that control S1P levels (Sphk1, sphingosine kinase 1; Sphk2, sphingosine kinase 2; Sgpl1, sphingosine-1-phosphate lyase; SGPP1, sphingosine-1-phosphate phosphatase 1; SGPP2, sphingosine-1-phosphate phosphatase 2; Spns2 (Spinster homolog 2) was performed by real-time qRT-PCR on intestinal tissues from mice and humans with IBD, compared with respective normal controls, (A) Patients with and without ulcerative colitis (n=8/group), (B) Patients with and without Crohn’s disease (n=8/group), (C) Mice treated with DSS or vehicle (H 2 O) (n=4/group), (D) Mice with (CD45RB Hi ) or without (CD45RB Hi+Lo ) T cell transfer colitis (n=4–6/group), (E) Uninflamed AKR and SAMP1/YitFc with spontaneous ileitis (n=7/group) and (F) Normal C57BL/6 and ileitic TNFΔARE (n=9/group). Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by two-tailed t test.

Article Snippet: Real-time qRT-PCR assays for SPHK1 (Hs00184211_m1), SPHK2 (Hs00219999_m1), SGPL1 (Hs00393700_m1), SGPP1 (Hs00229266_m1), SGPP2 (Hs00544786_m1), SPNS2 (Hs01390449_g1), Sphk1 (Mm00448841_g1), Sphk2 (Mm00445021_m1), Sgpl1 (Mm00486079_m1), Sgpp1 (Mm00473016_m1), Sgpp2 (Mm01158866_m1) and Spns2 (Mm01249324_m1) were performed using TaqMan Universal Master Mix (Applied Biosystems) with GAPDH/gapdh as endogenous controls.

Techniques: Expressing, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P deficiency enhances AA metabolism through p38-MAPK mediated cPLA 2 activation (A) The volcano plot illustrates the identification of 98 distinct metabolites between WT and Spns2 −/− PMs. AA and its derivatives are enriched in Spns2 −/− PMs. (B) KEGG enrichment analysis indicates elevated activities of glycerophospholipid metabolism and AA metabolism in Spns2 −/− PMs. (C) The heat map of the differential metabolites highlights the accumulation of AA derivatives and lysophospholipids in Spns2 −/− PMs. N = 6 biological replicates ( A to C ). ( D , E ) Deficient Spns2/S1P signaling enhances p38-MAPK mediated cPLA 2 activation. N = 3 biological replicates. Data are presented as mean ± s.e.m. (E) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons (E) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Altered AA metabolism promotes PGE 2 production in Spns2 −/− PMs (A) The flow diagram illustrating AA metabolism reveals a significant elevation in the gene expression of Ptges , encoding mPGES-1, in Spns2 −/− PMs. TPM, transcripts per kilobase of exon model per million mapped reads. (B) The volcano plot of up-regulated genes in Spns2 −/− PMs highlights the significant alteration in Ptges expression. N = 3 biological replicates ( A and B ). ( C ) Spns2 −/− PMs release elevated levels of PGE 2 under resting conditions. N = 5 biological replicates. Data are presented as mean ± s.e.m. ( A and C ). P values were determined by unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Gene Expression, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Overproduction of PGE 2 impairs MAS activity and mitochondrial dynamics (A) Gene expression of E-type prostanoid receptors in resting PMs. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Inhibition of EP4 with ONO-AE3-208, but not EP2 with PF-04418948, elevates the protein levels of MAS components Slc25a12 and Slc25a13 in Spns2 −/− PMs. N = 3 biological replicates. (C) EP4 activation contributes to the downregulation of Slc25a12 and Slc25a13 in WT PMs exposed to either PGE 2 or Spns2 inhibitor SLF1081851. N = 3 biological replicates. ( D , E ) Flow cytometry analysis of overlaid Δψm probed by MitoTracker™ Red (MT, D) and mitochondrial mass probed by CytoFix™ MitoRed (CF, E). MFI, mean fluorescent intensity. (F) Inhibition of EP4 restores the average Δψm (calculated by the ratio of MT/CF) in Spns2 −/− PMs. N = 3 biological replicates ( D to F) . (G) EP4 inhibition modulates the expression of mitochondrial dynamics-related proteins, promoting mitochondrial fusion in Spns2 −/− PMs. N = 3 biological replicates. (H) Transmission electron microscopy reveals that EP4 inhibition facilitates mitochondrial fusion in Spns2 −/− PMs. Scale bar = 1 μm. Arrowheads indicate fused (red) and fragmented (green) mitochondrial morphology. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to H) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activity Assay, Gene Expression, Inhibition, Activation Assay, Flow Cytometry, Expressing, Transmission Assay, Electron Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive EP4 activation impairs mitochondrial respiration and increases oxidative stress in Spns2 −/− PMs (A) Blocking EP4 with ONO-AE3-208 increases the oxygen consumption rates (OCR) in Spns2 −/− PMs. (B) Quantitative analysis of basal respiration, maximal respiration, ATP production, and proton leakage reveal the restoration of mitochondrial respiration following EP4 blockade. N = 4 biological replicates ( A and B) . (C) EP4 inhibition reduces intracellular lactate levels in Spns2 −/− PMs. N = 12 biological replicates. (D) Flow cytometry analysis reveals a decrease in MitoSOX™ Red-probed mtROS generation in ONO-AE3-208-treated Spns2 −/− PMs. N = 3 biological replicates. (E) Total intracellular ROS probed by CellROX ® Orange remains comparable among each group. N = 3 biological replicates. (F) EP4 inhibition diminishes the activities of total superoxide dismutase (SOD) and catalase, indicating alleviated oxidative stress in Spns2 −/− PMs. N = 6 biological replicates. Data in the panels A , B , D , and E are presented as mean ± s.e.m. In panels C and F , the central bands represent the median values, the boxes represent the distance between the third and the first quartile, and the whiskers represent the ranges between the minimum and maximum values. P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay, Blocking Assay, Inhibition, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: PGE 2 contributes to the early-phase hyperinflammation during bacterial infections ( A , B ) Flow cytometry analysis shows reduced mtROS generation probed by MitoSOX™ Red (A ) and decreased total intracellular ROS probed by CellROX ® Orange (B) in ONO-AE3-208-treated Spns2 −/− PMs at 3-h post-LPS challenge. N = 3 biological replicates ( A and B ). (C) EP4 blockade reduces the gene expression of inflammatory cytokines within 3-h post-LPS challenge due to the suppression of the lactate-ROS axis. Notably, EP2 blockade also attenuates the early-phase hyperinflammation, possibly via a mechanism independent of the lactate-ROS axis. Both EP2 and EP4 blockade partially restore the suppressed gene expression of inflammatory cytokines in Spns2 −/− PMs after 6-h post-LPS challenge. N = 3 biological replicates. (D) Schematic of the in vivo experiments using heat-killed E. coli -induced peritoneal infection models. (E, F) Both EP2 and EP4 inhibition alleviate hyperinflammation (E) and significantly improve survival rates (F) in Spns2 −/− sepsis models triggered by intraperitoneal infection with heat-killed E. coli . N = 6 biological replicates for cytokine measurement. N = 6 to 8 biological replicates for survival analysis. Data are presented as mean ± s.e.m. ( A , B , and E ) and percentage (F) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons ( A , B , and E ) and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Flow Cytometry, Gene Expression, In Vivo, Infection, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive PGE 2 production induces immunosuppression as infection progresses (A) Spns2 −/− PMs exhibit significantly elevated gene expression of Ptges compared to WT PMs before and after the LPS challenge. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Spns2 −/− PMs release higher levels of PGE 2 than WT PMs within 6-h post-LPS challenge. N = 6 biological replicates. (C) Gene expression of E-type prostanoid receptors in PMs at 3-h post-LPS challenge. N = 3 biological replicates. (D) Blockade of both EP2 and EP4 enhances TNFα and IL-6 release by Spns2 −/− PMs within 12-h post-LPS challenge. N = 4 biological replicates. (E) Schematic of the in vivo experiments using CLP models. (F) Survival curves from CLP models demonstrate that partial recovery of the inflammatory response induced by either EP2 or EP4 blockade improves the survival of Spns2 −/− rats. N = 7 to 12 biological replicates. (G) The levels of serum pro-inflammatory cytokines measured at 36-h post-infection indicate that EP2 or EP4 inhibition is effective but insufficient to overcome immunosuppression in Spns2 −/− CLP models. N = 4 biological replicates. (H) Colony-forming units (CFU) counts in livers and spleens at 36-h post-infection reveal higher bacterial loads in EP2- and EP4-inhibited Spns2 −/− CLP models. N = 5 to 6 biological replicates. Data are presented as mean ± s.e.m. ( A to D , G , and H ) and percentage (F) . P values were determined by unpaired t -test ( A to C ), one-way ANOVA with Sidak’s correction for multiple comparisons ( D , G , and H ), and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Infection, Gene Expression, In Vivo, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P signaling impacts mitochondrial functions through coordinated activation of multiple S1P receptors (A) All five S1PRs are transcriptionally detectable in PMs. N = 3 biological replicates. (B) Inhibition of individual S1PRs in WT PMs increases the gene expression of Ptges , while activation of S1PR2 and S1PR4 in Spns2 −/− PMs may slightly reduce Ptges expression. N = 4 biological replicates. (C) In WT PMs, blocking individual S1PRs reduces the fluorescence intensity of MitoTracker™ Red, indicating diminished ΔΨm. (D) S1PR3 blockade significantly increases the fluorescence intensity of CytoFix™ MitoRed, indicating increased mitochondrial mass, while blockade of other receptors may cause a slight increase in mitochondrial mass. (E) All treatments result in reduced average Δψm (calculated by the ratio of MT/CF) in WT PMs. N = 3 biological replicates ( C to E ). ( F , G ) In Spns2 −/− PMs, activation of S1PR3 and S1PR5 increases MitoTracker™ Red fluorescence intensity (F) , but none of the treatments affect mitochondrial mass (G) . (H) Only S1PR3 activation partially restores the average Δψm in Spns2 −/− PMs. N = 3 biological replicates ( F to H ). (I) All the antagonists increase mtROS generation in WT PMs, especially with S1PR2 and S1PR4 blockade. N = 3 biological replicates. (J) In Spns2 −/− PMs, activation of S1PR2 and S1PR4 attenuates oxidative stress. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to J ). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay, Inhibition, Gene Expression, Expressing, Blocking Assay, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Schematic illustration of how Spns2/S1P signaling modulates mitochondrial functions and inflammatory response through PGE 2 production in macrophages

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques:

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: CELF1 promotes circGlis3 formation in β cells (A) qRT-PCR analysis of the expression level of circGlis3 in MIN6 cells transfected with indicated siRNAs. (B) Schematic of Glis3 pre-mRNA showing the locations of four putative CELF1 binding sites (1–4) and amplicons (a–e) used for RIP assay. (C) MIN6 cells were transfected with or without wild-type (WT) circGlis3 minigene, and then RIP assays were performed. The relative RNA levels of CELF1-binding fragments in circGlis3-flanked introns were analyzed by qRT-PCR using the primers indicated in (B). IgG served as a control. (D) Western blot detected the protein levels of CELF1 in MIN6 cells transfected with Celf1 plasmid (ov- Celf1 ) or empty vector (Vector). (E) Schematic of circGlis3 minigenes with WT or mutant (Mut) CELF1 binding sites on the flanked intron regions of circGlis3. (F) MIN6 cells were stably transfected with Celf1 plasmid (ov- Celf1 ) or empty vector (Vector), and then transfected with WT or various mutant circGlis3 minigenes. qRT-PCR analyzed the expression level of circGlis3. (G) Western blot analyzed CELF1 protein levels in MIN6 cells treated with 0.3 mmol/L palmitate or 0.3% BSA for 24 h. (H) MIN6 cells with or without CELF1 knockdown were treated with 0.3 mmol/L palmitate, followed by qRT-PCR analysis of Celf1 and circGlis3 levels. (I) Western blot evaluated CELF1 protein levels in the islets of db / db and db / m mice (n = 4/group). (J) Correlation between circGlis3 and Celf1 in the islets of db / db and db / m mice was determined by qRT-PCR (n = 9/group). The statistical analysis was performed with Pearson’s correlation analysis. (K–M) MIN6 cells with or without CELF1 overexpression were transfected with shRNA targeting circGlis3 (sh-circGlis3) for rescue assays. Cell viability was detected by CCK-8 assay (K). Insulin secretion was assessed by GSIS assay (L). Western blot detected the expression levels of proteins (PCNA, Bcl-2 and Cleaved caspase-3) (M). The Western blots images are representative of three independent experiments unless otherwise noted. Data are represented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, by two-tailed Student’s t test in (C, D, G, and I), and one-way ANOVA (with Tukey post hoc test) in (A, F, H, and K–M).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Control, Western Blot, Plasmid Preparation, Mutagenesis, Stable Transfection, Knockdown, Over Expression, shRNA, CCK-8 Assay, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: CircGlis3 binds to hnRNPF and inhibits its nuclear translocation (A) Biotin-labeled circGlis3 probe (C-Glis3) and linear RNA probe with an identical sequence as circGlis3 (L-Glis3) were in vitro transcribed, then total protein of MIN6 cells was collected and RNA pulldown assay was performed. Flowchart illustrating the screening process for potential circGlis3-binding proteins. (B) Western blot detected hnRNPF levels in the samples pulled down by circular (C- Glis3 ) or linear (L- Glis3 ) RNA probe. (C) RIP assay was performed using anti-hnRNPF antibody, followed by qRT-PCR to detect circGlis3 enrichment. (D) Dual RNA-FISH and IF verified the colocalization of circGlis3 and hnRNPF in MIN6 cells. Scale bar, 10 μm. (E) Western blot analyzed the distribution of hnRNPF in the nucleus and cytoplasm of MIN6 cells with circGlis3 overexpression. GAPDH and Histone H3 were used as cytoplasmic and nuclear protein control, respectively (F) Schematic diagram showing the construction of Flag-tagged hnRNPF plasmids with full-length (FL) or different forms of truncation. (G and H) MIN6 cells were transfected with plasmids encoding Flag-tagged FL or truncated hnRNPFs. Western blot verified the expression of Flag-tagged recombinant hnRNPF protein (G). RIP-qPCR assay using anti-Flag antibody was performed to detect the enrichment of circGlis3 (H). (I) Schematic illustration of WT circGlis3 plasmid (circGlis3-WT) and circGlis3 plasmids with mutated RNA-binding motifs of hnRNPF within 526–577 bp (circGlis3-Mut1) or 851–902 bp (circGlis3-Mut2). (J) Dual RNA-FISH and IF analyzed the expression of circGlis3 and the distribution of hnRNPF in MIN6 cells transfected with the plasmids indicated in (I). Scale bar, 10 μm. (K–M) A mutant circGlis3 plasmid (circGlis3-Mut) was constructed by mutating the RNA-binding motifs of hnRNPF within 526–577 bp and 851–902 bp. MIN6 cells were transfected with circGlis3-WT or circGlis3-Mut plasmid. Cell viability was measured by CCK-8 assay (K). Insulin secretion was assessed by GSIS assay (L). Western blot detected the expression levels of PCNA and Bax (M). The Western blots images are representative of three independent experiments. Data are expressed as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ns indicates no statistical significance, by two-tailed Student’s t tests in (C, E, H), and one-way ANOVA (with Tukey post hoc test) in (K–M).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Translocation Assay, Labeling, Sequencing, In Vitro, Binding Assay, Western Blot, Quantitative RT-PCR, Over Expression, Control, Transfection, Expressing, Recombinant, Plasmid Preparation, RNA Binding Assay, Mutagenesis, Construct, CCK-8 Assay, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: CircGlis3 induces β-cell dysfunction by disrupting the hnRNPF/SIRT1 pathway (A–F) Scramble siRNA or siRNAs targeting hnRNPF (si- hnRNPF -1 and si- hnRNPF -2) were transfected into MIN6 cells for 48 h. Western blot detected the protein levels of hnRNPF (A). Cell viability was assessed by CCK-8 assay (B). Cell proliferation was determined by EdU staining. Scale bar, 100 μm (C). Cell apoptosis was evaluated by TUNEL staining. Scale bar, 100 μm (D). The expression levels of proteins (hnRNPF, PCNA, Bax and Cleaved caspase-3) were analyzed by Western blot (E). Insulin secretion was assessed by GSIS assay (F). (G) qRT-PCR analysis of the expression levels of circGlis3, Sirt1 , Ace2 , Nrf2 and Bmf in MIN6 cells with circGlis3 overexpression. (H) qRT-PCR detected the levels of hnRNPF and Sirt1 in MIN6 cells and primary islets with hnRNPF overexpression or knockdown. (I) Luciferase (Luc) activities of MIN6 cells co-transfected with luciferase reporter vector containing the Sirt1 gene promoter and pcDNA- hnRNPF plasmid or empty vector. Data were normalized by co-transfecting the Renilla luciferase (Rluc) reporter vector. (J) Luciferase activities of MIN6 cells co-transfected with plasmids containing various lengths of the Sirt1 gene promoter and hnRNPF overexpression vector or empty vector. (K–M) MIN6 cells with circGlis3 overexpression were transfected with pcDNA- hnRNPF plasmid (ov- hnRNPF ) or empty vector (Vector) for rescue assays. Cell viability was analyzed by CCK-8 assay (K). Insulin secretion was measured by GSIS assay (L). Western blot detected the protein levels of hnRNPF, SIRT1, PCNA and Cleaved caspase-3 (M). (N) qRT-PCR detected the levels of circGLIS3 in NES2Y cells treated with 0.3 mmol/L palmitate or 0.3% BSA for 24 h. (O and P) NES2Y cells were transfected with empty vector or circGLIS3 plasmid. Western blot analyzed the distribution of hnRNPF in the nucleus and cytoplasm of NES2Y cells (O). qRT-PCR measured the expression levels of SIRT1 in NES2Y cells (P). Values are represented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ns indicates no significance, by two-tailed Student’s t test in (G, J, N-P), and one-way ANOVA (with Tukey post hoc test) in (A–F, H, I, and K–M).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Transfection, Western Blot, CCK-8 Assay, Staining, TUNEL Assay, Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: CircGlis3 encodes a 348-amino acid (aa) protein (A) RIP assay was performed using anti-EIF2S1 antibody, followed by qRT-PCR to detect circGlis3 enrichment. (B–D) The polysome fractions of MIN6 cells were extracted using 10%–45% sucrose gradient centrifugation, and absorbance at 260 nm was measured. The relative levels of circGlis3 (B), Actin mRNA (C), and circPms1 (D) were analyzed by qRT-PCR in gradient fractions in MIN6 cell lysates. Actin and circPms1 served as positive and negative controls, respectively. (E) Top, the putative ORF, IRES and start/stop sites of circGlis3. Bottom, the amino acid sequences of Glis3-348aa encoded by the putative ORF, and specific amino acid sequences of Glis3-348aa are shown in red. The Glis3-348aa antibody was generated against the indicated sequences. (F) Schematic illustration of five types of plasmids used for luciferase reporter assays. WT, a series of deletions of IRES sequences (IRES-Del-1, IRES-Del-2) and mutated IRES sequences (IRES-Mut) were inserted into the vector, respectively. (G) The luciferase activity of Luc/Rluc was measured in the indicated five vectors. Luc, firefly luciferase; RLuc, renilla luciferase. (H) Schematic of the expression constructs. CircGlis3 sequence was inserted into a circular RNA expression vector containing Alu elements to form circGlis3 plasmid. FLAG tag was added directly to upstream of the stop codon (TAG) to establish the circGlis3-Flag plasmid. The circGlis3-Flag sequence was cloned to a linear vector to form a negative control plasmid (circGlis3-Flag-NC). (I and J) MIN6 cells were transfected with the indicated plasmids. The expression level of circGlis3 was analyzed by qRT-PCR (I), and the potential proteins were detected by Western blot (J). (K) Total protein lysates from MIN6 cells transfected with circGlis3 plasmid or empty vector were separated by SDS-PAGE, followed by Coomassie brilliant blue staining. The differential gel bands near 35 kDa were excised and subjected to mass spectrometry to identify the specific sequences of Glis3-348aa. (L) Western blot analysis of Glis3-348aa levels in MIN6 cells treated with BSA or palmitate. (M) Western blot evaluated the levels of CELF1 and Glis3-348aa in MIN6 cells with CELF1 overexpression. Results are expressed as mean ± SD of three independent experiments. ∗∗p < 0.01 and ∗∗∗p < 0.001, by two-tailed Student’s t test in (A, I, L, M), and one-way ANOVA (with Tukey post hoc test) in (G).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Quantitative RT-PCR, Gradient Centrifugation, Generated, Luciferase, Plasmid Preparation, Activity Assay, Expressing, Construct, Sequencing, RNA Expression, FLAG-tag, Clone Assay, Negative Control, Transfection, Western Blot, SDS Page, Staining, Mass Spectrometry, Over Expression, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: Glis3-348aa impairs β-cell function in vitro (A) Schematic illustration of the circGlis3-ORF-Flag and circGlis3-ORF-Flag-mut constructs. The circGlis3 ORF sequence with FLAG tag was inserted into a linear expression vector to form circGlis3-ORF-Flag. The stop codon TAG was deleted in the mutant construct, which failed to produce a protein. (B–H) MIN6 cells and primary mouse islets were transfected with the indicated plasmids. The expression levels of Flag and Glis3-348aa in MIN6 cells and islets were detected by Western blot (B). Cell proliferation in MIN6 cells (Scale bars represent 100 μm) and mouse islets (Scale bars represent 50 μm) was measured by EdU staining (C). Cell apoptosis in MIN6 cells (Scale bars represent 100 μm) and islets (Scale bars represent 50 μm) was evaluated by TUNEL staining (D). Cell viability of MIN6 cells was assessed by CCK-8 assay (E). Insulin secretion of MIN6 cells (F) and mouse islets (G) was measured by GSIS assay. Western blot analyzed the protein levels of PCNA, Bcl-2 and Cleaved caspase-3 in MIN6 cells and islets (H). Data are expressed as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, by one-way ANOVA (with Tukey post hoc test).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Cell Function Assay, In Vitro, Construct, Sequencing, FLAG-tag, Expressing, Plasmid Preparation, Mutagenesis, Transfection, Western Blot, Staining, TUNEL Assay, CCK-8 Assay

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: Glis3-348aa interacts with GLIS3 protein and attenuates its transcriptional activity (A) IF assay showed the colocalization of Glis3-348aa and GLIS3 in MIN6 cells transfected with HA-tagged GLIS3. Scale bar, 10 μm. (B) The interaction between Glis3-348aa and GLIS3 in MIN6 cells was analyzed by Co-IP assays using anti-Glis3-348aa antibody. (C, D) Co-IP assays confirmed the interaction between Glis3-348aa and GLIS3 in MIN6 cells with overexpression of HA-GLIS3 (C) or Flag-Glis3-348aa (D). (E) Schematic diagrams showing the construction of HA-tagged GLIS3 plasmids with full-length (FL) or different forms of truncation. (F) MIN6 cells were transfected with plasmids encoding HA-tagged FL or truncated GLIS3, followed by Co-IP with anti-HA antibody. (G–I) The circGlis3-ORF plasmid was generated by inserting circGlis3 ORF sequence into a linear expression vector. Luciferase plasmids containing the Ins2 , Ngn3 and Ccnd2 promoters were constructed, respectively. MIN6 cells with overexpression of circGlis3-ORF were co-transfected with indicated plasmids, followed by detecting the promoter activity of Ins2 (G), Ngn3 (H) and Ccnd2 (I) via dual-luciferase assays. (J–M) MIN6 cells with circGlis3-ORF overexpression were transfected with pcDNA- Glis3 plasmid (ov- Glis3 ) or empty vector (Vector). Western blot analyzed the protein levels of Glis3-348aa and GLIS3 (J). Cell viability was measured by CCK-8 assay (K). Insulin content was assessed by ELISA and normalized to total protein of cell lysates (L). Western blot detected the protein levels of CCND2, NGN3 and Cleaved caspase-3 (M). The Western blots images are representative of three independent experiments. Data are represented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, by two-tailed Student’s t test in (G-I), and one-way ANOVA (with Tukey post hoc test) in (J-M).

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Activity Assay, Transfection, Co-Immunoprecipitation Assay, Over Expression, Plasmid Preparation, Generated, Sequencing, Expressing, Luciferase, Construct, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet: Overexpression of circGlis3 promotes β-cell dysfunction in vivo (A) qRT-PCR analyzed the expression level of circGlis3 in the islets of Tg-circGlis3 mice and WT littermates (n = 4/group). (B and C) FBG levels (B) and IPGTTs (C) of 12-week-old Tg-circGlis3 and control mice ( n = 6–10/group). (D) Whole blood samples of 12-week-old Tg-circGlis3 and control mice were taken at 0, 15 and 30 min after glucose injection, and serum insulin levels were measured (n = 5/group). (E) IHC analysis of insulin and Ki67 expression in pancreatic sections from 14-week-old Tg-circGlis3 and control mice. Arrows indicate Ki67-positive β cells. Scale bar, 100 μm. (F) β cell apoptosis was evaluated by TUNEL (red) and insulin (green) coimmunostaining. Arrows indicate the DAPI/TUNEL/insulin copositive β cells. Scale bar, 50 μm. (G–I) The islets were isolated from 14-week-old Tg-circGlis3 and control mice. Western blot detected hnRNPF levels in the nucleus and cytoplasm of the islets (n = 4/group) (G). Western blot assessed the protein levels of SIRT1, hnRNPF and Glis3-348aa in the indicated mouse islets (n = 4/group) (H). qRT-PCR evaluated the expression levels of Ins2 , Ngn3 and Ccnd2 in the islets (n = 3/group) (I). (J–L) The islets were isolated from HFD-induced diabetic mice injected with AAV8-NC or AAV8-shcircGlis3. Western blot analyzed hnRNPF levels in the nucleus and cytoplasm of the islets (n = 3/group) (J). The protein levels of SIRT1 and Glis3-348aa in the islets were measured by Western blot (n = 3/group) (K). The expression levels of Ins2 , Ngn3 and Ccnd2 were evaluated by qRT-PCR (n = 3/group) (L). Data are shown as mean ± SD of independent experiment indicated as above. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ns indicates no significance, by two-tailed Student’s t test.

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Over Expression, In Vivo, Quantitative RT-PCR, Expressing, Control, Injection, TUNEL Assay, Isolation, Western Blot, Two Tailed Test

Journal: iScience

Article Title: circGlis3 promotes β-cell dysfunction by binding to heterogeneous nuclear ribonucleoprotein F and encoding Glis3-348aa protein

doi: 10.1016/j.isci.2023.108680

Figure Lengend Snippet:

Article Snippet: Mouse: circGlis3 fl/fl : C57BL/6J , Cyagen Biosciences , N/A.

Techniques: Virus, Recombinant, Modification, Lysis, Transfection, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Imaging, TUNEL Assay, Apoptosis Assay, In Situ Hybridization, Immunoprecipitation, Mass Spectrometry, Western Blot, shRNA, Plasmid Preparation, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: Spinster homolog 2 (SPNS2) expression is reduced in aging vascular and senescent endothelial cells (ECs) and has a negative correlation with aging degree. a Expression of SPNS2 in human carotid artery from GSE100927 (healthy carotid artery, n = 12, atherosclerosis carotid artery, n = 29, two-sided t -test). Source data are provided as a Source Data file. b , c mRNA levels of KI67 and Lamin B1 b and SPNS2 c in the aorta of mice during different months. Data are presented as the mean ± standard error of mean (SEM) ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. d Immunofluorescence of Lamin B1, SPNS2 and CD31 in the aorta of mice during different months. The cell nucleus exhibits blue fluorescence (DAPI), Scale bar, 100 μm. e Regression analysis of KI67 and LaminB1 expression data with SPNS2 expression from the aorta of mice during different months. f SA-β-gal staining of control human umbilical vein endothelial cell (HUVEC) and HUVECs treated with H 2 O 2 . Scale bar, 200 μm. g Immunoblot analysis of P53 and Lamin B1 in control HUVECs and HUVECs treated with H 2 O 2 . h Immunoblot analysis of SPNS2 in HUVECs and HUVECs treated with H 2 O 2 . i mRNA levels of SPNS2 in HUVECs and HUVECs treated with H 2 O 2 . Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test.

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency in mice ECs aggravates vascular dysfunction and collagen deposition. a, b Immunofluorescence of SPNS2, Lamin B1 and CD31 in the aorta of SPNS2 flox/flox and SPNS2 TEK−/− mice. The cell nucleus exhibits blue fluorescence (DAPI), Scale bar, 100 μm. c mRNA levels of SPNS2, KI67, Lamin B1, P21, MMP3, IL-6, IL-1β, and IL-8 in SPNS2 flox/flox and SPNS2 TEK−/− mice. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. d , e Small animal diagnostic ultrasound of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice. Blood flow analysis in SPNS2 flox/flox and SPNS2 TEK−/− mice. f Masson staining of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice quantified using ImageJ. Scale bar, 100 μm. No adjustments. g Sirius red staining of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice quantified using ImageJ. Scale bar, 100 μm. No adjustments

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Immunofluorescence, Fluorescence, Diagnostic Assay, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency aggravates HUVEC senescence. a Immunoblot analysis of SPNS2 in control, Sh-negative control (NC), and Sh-SPNS2 HUVECs. b Morphology of control, Sh-NC, and Sh-SPNS2 HUVECs by crystal violet staining and quantified by Image J. Scale bar, 200 μm. c The union of differential genes of all comparative combinations of Sh-NC and Sh-SPNS2 HUVECs. d Volcano plot showing fold changes in translation levels between Sh-NC and Sh-SPNS2 HUVECs. The upregulated (red) and downregulated (green) genes are highlighted. Source data are provided as a Source Data file. e KEGG analysis of the RNAseq data from HUVECs with SPNS2 knockdown. f SA-β-gal staining of control, Sh-NC, and Sh-SPNS2 HUVECs. Scale bar, 200 μm. g MTT assay detects cell viability of control, Sh-NC, and Sh-SPNS2 HUVECs. h Immunofluorescence of control, Sh-NC, and Sh-SPNS2 HUVECs. The cell nucleus exhibits blue fluorescence (DAPI), and KI67 exhibits yellow fluorescence (RFP). Scale bar, 200 μm. Enlarged image on the left. Scale bar, 50 μm. i Immunoblot analysis of Lamin B1 and P53 in control, Sh-NC, and Sh-SPNS2 HUVECs. j mRNA levels of Il-1β, Il-6, Il-8, and Mmp3 in control, Sh-NC, and Sh-SPNS2 HUVECs. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. k Immunoblot of SPNS2 in control, OE-negative control (NC), and OE-SPNS2 HUVECs. l mRNA levels of Il-1β, Il-6, Il-8, and Mmp3 in control, OE-NC, and OE-SPNS2 HUVECs. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t-test.

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Western Blot, Control, Negative Control, Staining, Knockdown, MTT Assay, Immunofluorescence, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency induces HUVEC senescence via mitochondrial function damage. a Transmission electron microscopic image of control, Sh-NC, and Sh-SPNS2 HUVECs. Mitochondria are highlighted by arrows, 500 nm. b Transmission electron microscopic image of control, Sh-NC, and Sh-SPNS2 HUVECs. Mitochondria are stained by Mitored (red), 10 μm. c Immunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in control, Sh- NC, and Sh-SPNS2 HUVECs d N, N, N′, N′-tetramethyl-ethylenediamine (TMRE) staining of control, Sh-NC, and Sh-SPNS2 HUVECs measured using flow cytometry. e DCFH-DA staining of control, Sh-NC, and Sh-SPNS2 HUVECs measured using flow cytometry. f TMRE staining of Sh-SPNS2 HUVECs and Sh-SPNS2 HUVECs treated with NAC measured using flow cytometry. g DCFH-DA staining of Sh-SPNS2 HUVECs and Sh-SPNS2 HUVECs treated with NAC measured using flow cytometry. h SA-β-gal staining of control, Sh-NC, Sh-SPNS2, and Sh-SPNS2 HUVECs treated with NAC. Scale bar, 200 μm. i I mmunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in control HUVECs and HUVECs treated with NAC. j Immunoblot analysis of Lamin B1 in HUVECs and HUVECs treated with NAC. k IL-6, IL-1β and IL-8 protein level of HUVECs and HUVECs treated with NAC were detected using ELISA kit

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Transmission Assay, Control, Staining, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency impairs mitochondrial function by promoting PKM-mediated pyruvate metabolism dysregulation. a Heatmap analysis shows the relative expression levels of pyruvate metabolism genes from GO enrichment. b Heatmap analysis shows the relative expression levels of HIF-1 signaling pathway genes from KEGG enrichment. c Immunoblot analysis of HIF1α in control, Sh-negative control (NC), and Sh-SPNS2 HUVECs. d , e , f , g , h Contents of pyruvate, ethanol, lactic acid, ATP, and acetyl Co-A in control, Sh-NC, and Sh-SPNS2 HUVECs. i Immunoblot analysis of PKM in control, Sh-NC, Sh-SPNS2 HUVECs, Sh-SPNS2 HUVECs treated with nc-siRNA, and Sh-SPNS2 HUVECs treated with PKM-siRNA. j , k , l Contents of pyruvate, ethanol, and lactic acid in Sh-SPNS2 HUVECs treated with nc-siRNA and those treated with PKM-siRNA. m Immunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in Sh-SPNS2 HUVECs treated with nc-siRNA and HUVECs treated with PKM-siRNA. n , o Contents of ATP and acetyl Co-A in Sh-SPNS2 HUVECs treated with nc-siRNA and Sh-SPNS2 HUVECs treated with PKM-siRNA

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Expressing, Western Blot, Control, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: Primers for qPCR

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: