Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The effect of FABP4 and M1-polarized macrophage supernatant on HUVECs, FLSs, and chondrocytes in vitro. a , b Tube formation assay and quantification of HUVECs cultured with vehicle (C), rhFABP4 (P), rhFABP4 + BMS309403 (PB), M1-polarized macrophage supernatant (S), or M1-polarized macrophage supernatant+BMS309403 (SB) ( n = 3 per group). Scale bar: 100 µm. c Immunoblot analysis of FABP4 and VEGFα in HUVECs cultured with C, P, PB, S, or SB for 24 h. d , g Representative images and quantification of BrdU (green) immunofluorescence ( d ) and Transwell assays ( g ) in HUVECs and FLSs treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). Scale bar: 25 µm, 100 µm. e , f Immunoblot analysis of ERK1/2, p-ERK1/2, P65, and p-P65 in HUVECs ( e ) and FLSs ( f ) treated with C, P, PB, S, or SB for 1 h. h Toluidine blue staining of ATDC5 cells treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). i Immunoblot analysis of FABP4, MMP13, Sox9, and Col2a1 in primary chondrocytes treated with C, P, PB, S, or SB for 24 h. j Immunoblot analysis of P65 and p-P65 in primary chondrocytes treated with C, P, PB, S, or SB for 1 h. One-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01 compared to the control. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: In Vitro, Tube Formation Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Comparison, Control

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Activation of the mTORC1 pathway enhances the secretion of FABP4 by M1-polarized macrophages to exacerbate RA progression. a , b Representative images and quantification of F4/80 and pS6 coimmunostaining in the mouse ( n = 10 per group) ( a ) and human synovium ( n = 16 per group) ( b ) of the control and RA groups. Scale bar: 12.5 µm. c Western blot showing S6 and pS6 in human synovial tissue. d Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with LPS or rapamycin. e Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs stimulated with LPS or rapamycin ( n = 3 per group). Scale bar: 100 µm. f Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with MHY1485 or rapamycin. g Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs treated with MHY1485 or rapamycin ( n = 3 per group). Scale bar: 100 µm. h Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs from control and TSC1KO mice. i Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of wild-type and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. j Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f–j Representative images ( f ) and quantification of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: Activation Assay, Control, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibition of the mTORC1 pathway reduces FABP4 secretion by M1-polarized macrophages to attenuate RA progression. a Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. b – e Representative images ( b ) and quantitative analysis of FABP4 immunohistochemical staining ( c ) and coimmunostaining of FABP4 with F4/80 ( d ) or NOS2 ( e ) in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f – j Representative images ( f ) and quantitative analysis of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: Inhibition, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Delivery of GLUT1 Inhibitor via Macrophage Nanovesicles for Pulmonary Arterial Hypertension Therapy

doi: 10.1002/jev2.70294

Figure Lengend Snippet: Mac@WZB117 reverses macrophage polarization imbalance in lung tissues of PAH mice. (A) Flow cytometry gating strategy: after FSC/SSC selection, F4/80 + was used as a macrophage marker, with CD86 and CD206 labelling M1 and M2 phenotypes, respectively. (B) The proportion of M1 macrophages decreased significantly to 9.2% ± 1.5%, then rose to 28.7% ± 2.6% after Mac@WZB117 intervention ( p < 0.01, n = 5). (C) The proportion of F4/80 + CD206 + M2 macrophages increased to 45.3% ± 3.7% in the PAH + NS group and decreased to 20.1% ± 2.2% after Mac@WZB117 treatment ( p < 0.01). Data are presented as mean ± SD. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: An in vitro M1 polarization model was established using the murine macrophage cell line RAW264.7 (ATCC, TIB‐71).

Techniques: Flow Cytometry, Selection, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Delivery of GLUT1 Inhibitor via Macrophage Nanovesicles for Pulmonary Arterial Hypertension Therapy

doi: 10.1002/jev2.70294

Figure Lengend Snippet: Glycolytic reactivation partially attenuates the therapeutic effects of Mac@WZB117. (A) Changes in macrophage phenotype proportions, FACS results showed an increased proportion of M2 macrophages and a decreased proportion of M1 macrophages in the rescue group, indicating reversal of macrophage polarization. (B) qPCR data showed iNOS expression was upregulated, while ARG1 and Mrc1 expression was downregulated, confirming that phenotype remodelling depends on glycolysis inhibition. (C) Haemodynamic parameters (RVSP and mPAP) were significantly elevated after rescue treatment, approaching levels observed in the PAH model. (D) Representative histological staining images and quantitative analysis showed that vascular WT% and α‐SMA‐positive area were markedly increased in the rescue group, suggesting partial reversal of pulmonary vascular structural remodelling. Data are presented as mean ± SD. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: An in vitro M1 polarization model was established using the murine macrophage cell line RAW264.7 (ATCC, TIB‐71).

Techniques: Expressing, Inhibition, Staining

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: PATJ mRNA expression determined by RT-PCR ( A , C , D ) and by array-based gene expression ( B ). A Lower PATJ mRNA expression in peripheral blood extracted 24 h after IS in patients with functional independence (mRS < 3, n = 25) compared with patients with marked disability (mRS > 3, n = 25). * p < 0.05 as compared to mRS > 3 (Student’s t-test). B The G risk allele of rs76221407 SNP of PATJ gene confers higher expression in peripheral blood. * p < 0.05 as compared to the AA genotype (Student’s t-test). C Patj expression significantly decay in the ischemic region of mice brain subjected to 1 h transient middle cerebral artery occlusion. Patj mRNA levels were compared between the ipsilateral ischemic region (IL) and the contralateral CTL region (CL) of cortical brain homogenates, measured at 48 h after reperfusion. ** p < 0.01 as compared to CL region (Paired t-test). D Oxygen-glucose deprivation (OGD) for 5 and 10 h induces PATJ downregulation in hCMEC/D3 ECs. ** p < 0.01; * p < 0.05 as compared to normoxia (Student’s t-test). E PATJ steady-state levels significantly decrease in ECs subjected to HIF-1α-dependent chemical hypoxia. Depletion of the 80 kDa specie of PATJ observed after 4 h incubation with cobalt chloride (CoCl 2 ). Prolonged incubation for 24 and 48 h accounted for depletion of the 230 and 200 kDa species. GAPDH was used as loading control. F Relative quantification of the integrated band intensities of three different WBs. Bars represent mean intensity ± SD. * p < 0.05; ** p < 0.01 as compared to CTL cells (ANOVA followed by Bonferroni’s test).

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Functional Assay, Incubation, Control, Quantitative Proteomics

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: A WB showing PATJ expression in three clones. Five main species were identified (230, 200, 135, 80, and 60 kDa). GAPDH was used as loading control. B Relative quantification of the integrated band intensities of three different WBs for PATJ species. Bars represent mean intensity ± SD. * p < 0.05; ** p < 0.01; *** p < 0.005 and **** p < 0.0001 as compared to CTL cells (ANOVA followed by Bonferroni’s test). C PATJ KD3 cells lost their cell-cell interactions, as evidenced by immunofluorescence against the TJs protein zonula occludens 1 (ZO-1). D Differential expression of TJs proteins in the three PATJ KD cells. WB analyses showed that the loss of TJs in PATJ KD3 was due to the depletion of its constituent proteins ZO-1, occludin, and claudin-11. The two intermediate PATJ KD clones (KD1 and KD2) had different immunoreactive bands for occludin and ZO-1 than CTLs. E Transwell permeabilization assay evidenced a complete loss of permeability of the PATJ KD3 cells. * p < 0.05 as compared to CTLs (Student’s t-test).

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Expressing, Clone Assay, Control, Quantitative Proteomics, Immunofluorescence, Permeability

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: A Hierarchical cluster showing the 74 most differentially expressed genes between PATJ KD and CTLs in the normalized (Log2 fold change (FC)) heatmap. PATJ KD1, 2, 3, and 4 were used. Level of expression was represented by color scale from green (low) to red (high), as indicated by the scale bar at the top. B Gene ontology (GO) analysis among the significantly modulated genes with an FDR cut-off of 0.05 revealed enrichment of biological processes such as Endothelial to Mesenchymal Transition (EndMT), Unfolded Protein Response (UPR) and Actin Cytoskeleton Organization. The results are visualized as a dot plot with an X-axis representing the gene ratio and the Y axis representing the FDR p -value. C Gene Set Enrichment Analysis (GSEA) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment between PATJ KD and CTL cells identified Notch, BMP and G-protein coupled receptor signaling as significant signaling pathways. D EnhancedVolcano plot of −log10 ( p -values) vs. log2 FC. The −log10 ( p -values) represents the level of significance of each gene while log2 FC represents the difference between the levels of expression for each gene between PATJ KD and CTL cells. E Gene-concept network (Cnetplot) showing the links between genes and the most significant cell signaling pathways, obtained with an FDR cut-off of 0.05, by using GSEA WikiPathways. The size of the concept nodes depends on the gene count involved in that pathway, while the color of the gene nodes depends on their expression level according to the displayed color gradient representing the log2 FC. Each number represents a significant pathway: NOTCH (1); BMP (2); TFG- β (3); PI3K/Akt (4) and VEGFA-VEGFR (5).

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Expressing, Protein-Protein interactions

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: A Cytosolic (Cyt) and Nuclear (Nucl) localization of ZEB1, β-catenin, YAP/TAZ, and MUC1-C. Voltage-dependent anion channel (VDAC) and LIM Kinase (LIMK) were used as cytosolic markers and Histone 3 (H3) as nuclear marker. B – F Quantifications of nuclear/cytosolic ratios for YAP ( B ), TAZ ( C ), MUC1-C ( D ), β-catenin ( E ) and ZEB1 ( F ). G Immunofluorescence using phalloidin to label F-actin (red) and YAP-antibody (green). H , I PATJ KD decreased the F/G actin ratio. H Representative immunoblot of F-actin and G-actin. I Relative quantification of the F/G actin ratio. Bars represent mean intensity ± SD of three independent experiments. *** p < 0.005 as compared to CTL cells (ANOVA followed by Bonferroni’s test). J WB analysis showing depletion of the actin cytoskeleton markers myosin light chain (MLC) and ARHGAP6 in PATJ KD3 cells.

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Marker, Immunofluorescence, Western Blot, Quantitative Proteomics

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: A Differential expression of the cell adhesion molecules ICAM-1 and CD44, as well as TRAF6 in PATJ KD cells, revealed by WB analysis. B , C Basal secretion of IL-6 ( B ) and IL-8 ( C ). PATJ KD3 cells produce significantly higher amounts of IL6. D , E Secretion of IL-6 ( D ) and IL-8 ( E ) after LPS incubation. Bars represent the mean of media cytokine detection (pg/ml) ± SD of three independent experiments. Counts were performed after 24 h of cell culture ( B , C ) or after 2, 4, or 8 h incubation with LPS ( D , E ). * p < 0.05, as compared to CTL cells (ANOVA followed by Bonferroni’s test).

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Quantitative Proteomics, Incubation, Cell Culture

Journal: Cell Death Discovery

Article Title: Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis

doi: 10.1038/s41420-024-01857-z

Figure Lengend Snippet: A , B Wound healing assay showed that intermediate PATJ KD clones migrate significantly faster after 4 and 8 h post-scratch. C , D Intermediate PATJ KD clones retain the ability for tubulogenesis, meanwhile the PATJ KD3 with mesenchymal phenotype could not perform angiogenesis in vitro, as shown with the tubular network formation assay of cells grown on Matrigel (10 mg/ml). D Total tube length quantification of three independent experiments, measured with the customized computer analysis “Angiogenesis Analyzer” for the Image J Program. * p < 0.05; ** p < 0.01 and **** p < 0.001 as compared to CTL cells (ANOVA followed by Bonferroni’s test).

Article Snippet: The RNA samples from the individuals of the GRECOS project [ ] were extracted from isolated buffy coat using the RiboPureTM-Blood kits (Ambion, Woodward St. Austin, USA). qPCR on these samples was performed on a 7900 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using a TaqMan fluorogenic probe for PATJ (Hs00902482_m1) and normalizing with Cyclophilin A (PPIA, Hs99999904_m1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, Hs99999905_m1).

Techniques: Wound Healing Assay, Clone Assay, In Vitro, Tube Formation Assay

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Determination of the molecular mechanism of WISP1v1 on the cell invasion in the bladder cancer cells. (A) The protein levels of NDRG1, KAI1, and Maspin of HT-DNA and HT-WISP1v1 cells were determined by immunoblot assays. (B) The luciferase activity of the NDRG1 reporter vector was shown after transiently overexpressing various amounts of WISP1v1 expression vectors in HT1376 and T24 cells, as indicated. (C) The luciferase activity of KAI1 and Maspin reporter vectors was shown as indicated after transiently overexpressing various dosages of WISP1v1 expression vectors in HT1376 cells. (D) The luciferase activity of NDRG1, KAI1, and Maspin reporter vectors, respectively, after transiently overexpressed pcDNA, WISP1v1, and WISP1v2 expression vectors as indicated in HT1376 cells. (E) The mRNA levels of the E-cadherin, N-cadherin, Slug, Snail, and Vimentin were determined by RT-qPCR. Data are presented as target genes/β-actin of HT-WISP1v1 cells relative to HT-DNA cells. The mRNA levels of the WISP1 (F), NDRG1, KAI1, Maspin, and IL-6 (G) after transient overexpression of WISP1v1 in T24 cells were determined by RT-qPCR. Data are presented as target genes/β-actin of T24-WISP1v1 cells relative to T24-DNA cells. (H) The invasion ability of T24 cells after the ectopic overexpression of WISP1v1 was determined by in vitro Matrigel invasion assays (± SE; n = 4). The ability of the migration (I) and quantification analysis (I) of T24 cells after being treated with the supernatant from 293T-DNA and 293T-WISP1v1 cells was determined by wound healing assays. The white line indicated the average of the leading cellular edges, and the wound area size was calculated by Image J. * p < 0.05, ** p < 0.01.

Article Snippet: TaqManTM gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), CXCL5 (Hs01099660_g1), SDF-1/CXCL12 (Hs03676656_m1), NDRG1 (Hs00608387_m1), KAI1 (Hs00356310_m1), Maspin (Hs00985283_m1), E-cadherin (Hs01023894_m1), N-cadherin (Hs00169953_m1), Snail (Hs00195591_m1), Slug (Hs00161904_m1), Vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania).

Techniques: Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, In Vitro, Migration

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Modulating effect of WISP1v1 on the cell growth of human bladder cancer HT1376 cells in xenograft animal model. (A) HT-DNA and HT-WISP1v1 cells were xenografted subcutaneously in the athymic nude mice ( n = 5). (B) The body weight of xenografted mice was measured as indicated during the experimental period. (C, top) Tumors from the HT-DNA and HT-WISP1v1 cells were recorded after the mice were sacrificed. The volumes of tumors were recorded as indicated during the experimental period (C, bottom). (D) The tumor weights of each group were recorded after the sacrifices. (E) The mRNA levels (± SE; n = 3) of WISP1v1, NDRG1, KAI1, and Maspin were determined by RT-qPCR. (F) The relative protein levels of WISP1v1, NDRG1, KAI1, Maspin, and β-actin (± SE) of the tumors from the HT-DNA and HT-WISP1v1 groups were determined by immunoblot assays. The quantitative analysis was presented as the relative density of the target proteins/β-actin relative to sample #1. * p < 0.05, ** p < 0.01.

Article Snippet: TaqManTM gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), CXCL5 (Hs01099660_g1), SDF-1/CXCL12 (Hs03676656_m1), NDRG1 (Hs00608387_m1), KAI1 (Hs00356310_m1), Maspin (Hs00985283_m1), E-cadherin (Hs01023894_m1), N-cadherin (Hs00169953_m1), Snail (Hs00195591_m1), Slug (Hs00161904_m1), Vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania).

Techniques: Animal Model, Quantitative RT-PCR, Western Blot