Journal: Nature

Article Title: Reduced cyclin D3 expression in erythroid cells protects against malaria

doi: 10.1038/s41586-026-10110-9

Figure Lengend Snippet: a , Top, vector content and expression of luciferase reporter genes driven by the extreme allele combinations: on the one hand the WT rs9349205-G|rs112233623-C (G|C) alleles on the other hand the derived rs9349205-A| rs112233623-T (A|T) alleles of the CCND3 enhancer in the erythroid cell line HUDEP-2, with empty vector as a control construct. Below, histograms show averages of the relative luminescence activity of the combinations of the two extreme alleles at rs9349205 and rs112233623 described above in absence (-) or presence of GATA1 and FOG-expressing plasmids; SMAD3 and p300 expressing plasmids; SMAD3 expressing plasmid alone; or empty expression vector. All values are plotted relative to the WT (G|C) construct. Note that the activity of the empty pGL4 vector is barely detectable. The mean ± s.e.m is shown (n = 7 (vectors); n = 3 (vectors + SMAD3), n = 3 (vectors+SMAD3 + p300), n = 4 (vectors + GATA1 + FOG) biologically independent experiments). A two-sided Student’s t -test was used, with level of significance indicated by asterisks (*P < .05;**P < .01; ****P < .0001, ns, not significant). The statistical results for all comparisons are provided in Supplementary Table . b , In silico prediction of binding to rs112233623 allele variants, showing derived allele T hindering binding of SMAD3 while favouring GATA1 binding. c , d , Representative electrophoretic mobility shift assays (EMSA) showing binding of SMAD3 and GATA1 proteins with labelled oligonucleotide probes (*) containing the WT (C) or derived (T) allele of rs112233623 (n = 3 biologically independent experiments). Competitor unlabelled oligonucleotides were used at the indicated fold excess to demonstrate specificity of binding. c , SMAD3 binds to the WT rs112233623-C allele (lanes 2 and 8); is weakly supershifted by anti-SMAD3 antibody (lanes 3 and 9); and is competed away by an excess of unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 10-12, at 25X, 50X and 100X respectively), but not by an excess of oligonucleotide containing the derived rs112233623-T allele (lanes 13-15, at 25X, 50X and 100X respectively). d , GATA1 binds to the rs112233623-T derived allele (lanes 2, 8 and 9), is supershifted by anti-GATA1 antibody (lane 3); and is competed away by an excess of unlabelled DNA oligonucleotides containing the derived rs112233623-T allele (lanes 10-12, at 25X, 50X and 100X respectively) compared to unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 13-15, at 25X, 50X and 100X respectively). GATA1 does not appear to bind to the WT rs112233623-C allele (lane 5). The samples derive from the same experiment and, the gels were processed in parallel. For gel source data, see Supplementary Fig. . e , f , ChIP–qPCR for SMAD3 or GATA1 binding to the CCND3 enhancer region surrounding rs112233623 ( CCND3 ) and to a SimpleChIP Human α Satellite as negative control in erythroblasts derived from individuals homozygous for the rs112233623-T decrease of expression (DoE) allele versus the WT rs112233623-C allele (WT). e , ChIP was performed using an antibody against SMAD3, results are represented as percentage of input and nonspecific IgG used as negative control. The mean ± s.e.m is shown (n = 3 biologically independent experiments). A two-sided two-Sample t-test was used; significant differences are indicated (*P < .05; ns, not significant). The statistical results for the comparisons are provided in Supplementary Table . f , ChIP assays were conducted with an antibody against GATA1, results are represented as percentage of input and nonspecific IgG used as negative control (n = 2 biologically independent experiments).

Article Snippet: ChIP DNA was purified and subsequently quantified by qPCR using primers designed to amplify a region surrounding rs112233623 in the CCND3 enhancer and SimpleChIP Human α Satellite Repeat Primers (4486, Cell Signaling Technology).

Techniques: Plasmid Preparation, Expressing, Luciferase, Derivative Assay, Control, Construct, Activity Assay, In Silico, Binding Assay, Electrophoretic Mobility Shift Assay, ChIP-qPCR, Negative Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 promotes colorectal cancer metastasis through a positive feedback loop mediated by CAF-secreted CXCL12. A, Mass spectrometry analyzed supernatants from SPP1-stimulated and unstimulated CAFs, showing fold changes in secreted proteins (SPP1/control). B, A bubble chart displays commonly secreted protein levels in fibroblasts. C and D, Uniform Manifold Approximation and Projection (UMAP) plots and quantitative analysis reveal CXCL12 expression in fibroblasts within OE-SPP1 and vector groups. E, ELISA measured CXCL12 in CAF supernatants with/without SPP1 (1 µg/mL), n = 3. F, A flowchart shows CAF-conditioned medium’s (CM) impact on colorectal cancer (CRC) cell migration and invasion. G and H, Transwell and wound healing assays evaluated the effects of CAF-conditioned media or CXCL12-neutralizing antibody (100 ng/mL) on colorectal cancer cell migration and invasion ( n = 3). I–K, Flowchart illustrating the effects of CXCL12 or neutralizing antibody treatment on the colorectal cancer cell migration and invasion, assessed via transwell and wound healing assays ( n = 3). L, The effect of CXCL12 (100 ng/mL) or a neutralizing antibody (100 ng/mL) on the epithelial–mesenchymal transition markers expression in the colorectal cancer cells was analyzed using Western blotting ( n = 3). M, Correlation analysis of CXCL12 with SPP1 and TGFB1 in the TCGA dataset. N and O, The effect of CXCL12 (100 ng/mL) or neutralizing antibody (100 ng/mL) on the SPP1 and TGFβ expression in the colorectal cancer cells was evaluated using Western blotting ( N ) or ELISA ( O ), n = 3. Results are presented as mean ± SEM. P values were calculated using a two-tailed unpaired Student t test ( E ), whereas one-way ANOVA was used for the other comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Mass Spectrometry, Control, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Migration, Western Blot, Two Tailed Test

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 inhibits T-cell infiltration and cytotoxicity via CXCL12 secretion from CAFs. A, Schematic of the coculture system with PDOs, T cells, and CAFs. CRC, colorectal cancer; E:T, effector to target. B and C, Confocal microscopy assessing the effect of SPP1 overexpression on T-cell infiltration and cytotoxicity in PDOs with or without CAFs ( n = 3). D and E, Impact of rhSPP1 (1 µg/mL) or CXCL12-neutralizing antibody (100 ng/mL) on T-cell infiltration and cytotoxicity in PDOs ( n = 3). Results are presented as mean ± SEM. P values were determined using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant. PI, propidium iodide.

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Confocal Microscopy, Over Expression

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 activates the β-catenin/HIF1α axis in the CAFs to drive CXCL12 secretion. A, Western blotting assessed key signaling pathway in CAFs after 24 hours of SPP1 protein stimulation. B–E, β-catenin and HIF1α expressions were analyzed following SPP1 or conditioned medium treatments, including from SPP1-overexpressing or -knockdown cells. F–H, HIF1α degradation was evaluated with MSAB or si-CTNNB1 transfection after cycloheximide (CHX) treatment, and HIF1α levels were measured after MSAB (1 µmol/L) or MG132 (20 µmol/L) pretreatment. I and J, Coimmunoprecipitation examined the HIF1α and β-catenin interaction. K and L, Immunofluorescence and nuclear–cytoplasmic fractionation assays assessed HIF1α and β-catenin localization ( n = 3). Scale bar, 25 μm. M–O, CXCL12 levels in conditioned media were measured after SPP1 (1 µg/mL) or MSAB treatments (24 hours). P, Correlation analysis of HIF1α and CXCL12 expression in 50 CAF samples using transcriptome data. Q, Dual-luciferase assays evaluated CXCL12 promoter activity ( n = 3). R and S, T-cell migration and infiltration were analyzed with or without SPP1 protein or MSAB treatment, n = 3. Scale bar, 50 μm. Western blotting ( A–J and L ) and ELISA ( M–O ) were repeated three times, with data representative of three independent experiments. Results are presented as mean ± SEM. P values were determined by one-way ANOVA ( M –O , R , and S ) and two-tailed unpaired Student t test ( F , G , and Q ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. R and S , Created with Figdraw.com .

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Western Blot, Knockdown, Transfection, Immunofluorescence, Fractionation, Expressing, Luciferase, Activity Assay, Migration, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: Blocking the SPP1/CXCL12 axis alleviates immunosuppression in the liver microenvironment and augments the benefits of immunotherapy. A, Flowchart of the intrasplenic injection model of liver metastasis using OE-SPP1 MC38 cells ( i.s.v. , intrasplenic injection; i.p. , intraperitoneal injection). B–D, Representative tumor morphology, hematoxylin and eosin staining, liver weight, and tumor burden ( n = 5 mice/group). Scale bar, 1 mm. E and F, Flow cytometric analysis of IFNγ + CD8 + and GZMB + CD8 + T cells in liver metastases ( n = 5 mice/group). G, Flowchart of the cecal orthotopic injection model of liver metastasis in the NOG mice using HCT116-HM cells. H and I, Luciferase images and bioluminescence quantification of metastatic livers. J, Hematoxylin and eosin staining and the number of liver metastases ( n = 5 mice/group). K, ELISA analysis of IFNγ levels in liver metastases ( n = 5 mice/group). L–N, ELISA of SPP1 and CXCL12 in peripheral blood of responders ( n = 25) and nonresponders ( n = 12) in immunotherapy-treated colorectal cancer cohorts. O, Diagram of tumor-derived SPP1 activation of CAFs to promote immunotherapy resistance in CRLM. Data are presented as mean ± SEM. P values were determined using one-way ANOVA ( C–F , and I–K ) and two-tailed unpaired Student t test ( L and M ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. O, Created in BioRender. Liu, F. (2025) https://BioRender.com/k7tx8am .

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Blocking Assay, Injection, Staining, Luciferase, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay, Two Tailed Test

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role and Therapeutic Potential for Targeting Fibroblast Growth Factor 10/FGFR1 in Relapsed Rheumatoid Arthritis.

doi: 10.1002/art.42674

Figure Lengend Snippet: Figure 2. scRNA-seq and ST reveal the heterogeneity of FLSs in relapse and Rem RA. (A) tSNE plots show the four FLS subsets in relapse and Rem RA, color coded according to cell subsets, including FB L-CD55 and three sublining subsets, FB S-CXCL12, FB S-VEGFA, and FB S-MFAP5. (B) Histogram shows the proportion of FLS subsets across samples. (C) Bubble plot of the subset-specific markers (top 10) in FLSs is given, with color representing the scaled average expression and sized by the fraction of cells (average expression >0). (D) Heatmap displays scaled expression values of the top DEGs of each subset of FLSs, grouped by relapse and remission RA. (E) Heatmap to show the enriched pathways (top five) of four FLS subsets in relapse and Rem RA according to the Hallmark and Reactome data sets. (F) Violin plots (right) show the enrichment scores of selected pathways of FLS subsets in relapse and Rem RA. Heatmap (left) shows the average expression of selected genes from enrichment path- ways as in paired violin plots. (G) The selected pathways enrichments scores in paired ST samples. (H) mIHC shows PDPN+IFI6+ FLSs located in the lining layer of the pannus in relapse RA. The white arrows represent the PDPN+IFI6+ FLSs. The positive area proportion of IFI6 expression of pannus in relapse and remission RA by IHC. ****P < 0.0001. Act-P, pannus of relapse RA; Act-S, synovium of relapse RA; DAPI, 4’,6-diamidino-2-phenylin- dole; DEG, differentially expression gene; FB L-CD55, CD55+ lining fibroblasts; FB S-CXCL12, CXCL12+ fibroblasts; FB S-MFAP5, MFAP5+ fibro- blasts; FB S-VEGFA, VEGFA+ fibroblasts; FLS, fibroblast-like synoviocyte; IFI6, interferon alpha–inducible protein 6; IL, interleukin; JAK, Janus kinase; MFAP5, microfibril associated protein 5; MMP, matrix metalloproteinase; mIHC, multiplex immunohistochemistry; NF-κB, nuclear factor-κB; PDPN, podoplanin; RA, rheumatoid arthritis; Rem, remission; Rem-S, synovium of remission RA; scRNA-seq, single-cell RNA sequencing; ST, spa- tial transcriptomics; STAT, signal transducer and activator of translation; tSNE, t-distributed stochastic neighbor embedding. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42674/abstract.

Article Snippet: The MH7A FLS cell line was obtained from iCell Bioscience (China) and was stimulated with recombinant human interferon alpha 2a (CST; catalog no. 93613S) or FGF10 protein (Peprotech; catalog no.100-26) for six hours, and qRT-PCR measured the target molecules.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Multiplex Assay, Immunohistochemistry, RNA Sequencing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role and Therapeutic Potential for Targeting Fibroblast Growth Factor 10/FGFR1 in Relapsed Rheumatoid Arthritis.

doi: 10.1002/art.42674

Figure Lengend Snippet: Figure 4. IFN-α and FGFR1 signaling pathways independently regulate the FLSs of relapse RA. (A and B) Correlation analysis between the IFN-α and FGFR1 signaling pathways in FLSs (Spearman’s correlation) with 95% confidence. (C) The IFI6, ISG15, and FGF10 mRNA levels in IFN-α2a–stimulated MH7A synovial fibroblast cell line quantified by real-time qPCR are shown. Values represent at least three independent experiments’ mean (±SD). ns, not significant; **P < 0.01; ****P < 0.0001. (D) The mRNA levels of IFI6 and ISG15 in the FGF10-stimulated MH7A synovial fibroblast cell line quantified by qPCR are shown. Values represent the mean (±SD) of three independent experiments. FGF, fibroblast growth factor; FGFR, FGF receptor; FLS, fibroblast-like synoviocyte; IFI6, IFN-α–inducible protein 6; IFN-α, interferon alpha; ISG15, interferon-stimulated gene 15; mRNA, messenger RNA; NC, untreated control group; qPCR, quantitative PCR; RA, rheumatoid arthritis. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42674/abstract.

Article Snippet: The MH7A FLS cell line was obtained from iCell Bioscience (China) and was stimulated with recombinant human interferon alpha 2a (CST; catalog no. 93613S) or FGF10 protein (Peprotech; catalog no.100-26) for six hours, and qRT-PCR measured the target molecules.

Techniques: Protein-Protein interactions, Control, Real-time Polymerase Chain Reaction