Journal: Journal of Translational Medicine

Article Title: Comprehensive investigation of malignant epithelial cell-related genes in clear cell renal cell carcinoma: development of a prognostic signature and exploration of tumor microenvironment interactions

doi: 10.1186/s12967-024-05426-x

Figure Lengend Snippet: Intertumoral heterogeneity of tumor epithelial cells in ccRCC. ( A ) UMAPs of all malignant epithelial single-cell transcriptomes color-coded by cell types. ( B ) Violin plot of the MECRGS scores in different cancer cell clusters. ( C ) Box plot showing CytoTRACE scores among cancer cells. ( D ) CNV scores revealing the differences between different malignant cell clusters. ( E ) Multicolor immunofluorescence staining of CA9, PLOD2 and SAA1 in the Renji TMA cohort. Scale bar: 50 μm. Color: CA9 (pink), PLOD2 (red), SAA1 (green), and cell nucleus (blue). Arrow: PLOD2 + SAA1 + CA9 + cells. ( F - I ) Feature plots of PLOD2, SAA1, C1S, C1R (left); The PLOD2, SAA1, C1S, C1R expression levels were positively related with tumor stages (right). ( J - M ) IHC images of C1S, C1R, PLOD2, and SAA1 between low and high pathology grades in the Renji TMA cohort. Scale bar: 100 μm. Left: patients with low pathology grades; right: patients with high pathology grades. ( N ) Gene ontology enrichment of all components of cancer cells

Article Snippet: The protocol involved the following steps: First, the TMA sections were incubated with primary antibodies against Anti-PLOD2 antibody (rabbit, 21214-1-AP, Proteintech); Anti-SAA1 antibody (rabbit, A1655, Abclonal); Anti-C1S antibody (rabbit, 14554-1-AP, ProteinTech); Anti-C1R antibody (rabbit, A25032, Abclonal).

Techniques: Multicolor Immunofluorescence Staining, Expressing

Journal: Journal of Translational Medicine

Article Title: Comprehensive investigation of malignant epithelial cell-related genes in clear cell renal cell carcinoma: development of a prognostic signature and exploration of tumor microenvironment interactions

doi: 10.1186/s12967-024-05426-x

Figure Lengend Snippet: Exploration of the C3-C3AR1 pairs and the significance of MECRGS in immunotherapy cohort. ( A ) The communications between PLOD2 + SAA1 + cancer cells and other cell types through complement signaling pathway. ( B , C ) IHC images of C3 between low and high pathology grades in the Renji TMA cohort. Scale bar: 100 μm. ( D ) Multicolor IF images of CD68, CD163 and FLOX3 between low and high pathology grades in the Renji TMA cohort. Scale bar: 100 μm. CD68 + cell (pink), CD163 + cell (red), FLOX3 + cell (green), and cell nucleus (blue). ( E ) Multicolor IF images of CA9, PLOD2 and SAA1 between low and high pathology grades in the Renji TMA cohort. Scale bar: 100 μm. CA9 + cell (pink), PLOD2 + cell (red), SAA1 + cell (green), and cell nucleus (blue). ( F ) Kaplan– Meier survival analysis of low- and high- MECRGS groups in CheckMate025 cohort. ( G ) Umap plot demonstrating the distribution of three tumor cell types. ( H ) Violin plot of the MECRGS scores in different cancer cell clusters. ( I ) Dot plots illustrating the distribution of marker genes across three tumor cell clusters. ( J ) Heatmap plot showing the expression of immune checkpoints across three tumor cell clusters. ( K ) Bar plot showing cell population distributions across PR, NR and NoICB samples. ( L ) Volcano plot showing the DEGs between NR and PR patients after immunotherapy

Article Snippet: The protocol involved the following steps: First, the TMA sections were incubated with primary antibodies against Anti-PLOD2 antibody (rabbit, 21214-1-AP, Proteintech); Anti-SAA1 antibody (rabbit, A1655, Abclonal); Anti-C1S antibody (rabbit, 14554-1-AP, ProteinTech); Anti-C1R antibody (rabbit, A25032, Abclonal).

Techniques: Marker, Expressing

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: Overall survival in TCL patients with and without prognostic factors—comparison of the Kaplan–Meier survival curves and prognostic factors, positive/negative immunostaining between Group 2 and Group 3 (log-rank test). ( A , B ) show the results of comparisons among 3 groups, and C to F show the results of comparisons between 2 groups. ( A ) GRP94+, CR1+, n = 2; GRP94+, CR−, n = 8; GRP94−, CR+, n = 6; p < 0.01. GRP94-positive patients showed a non-CR treatment response, resulting in a poor prognosis. ( B ) GRP94-positive patients with relapse ( n = 10), poor prognosis with a median OS of 13.5 months ( p < 0.01). GRP94-negative patients without relapse ( n = 6), relatively good prognosis with a median OS of 102 months ( p < 0.01). A total of 10 patients, consisting of 6 patients who showed a non-CR treatment response after the initial therapy and 4 patients who developed relapse within 1 year, were identical to the 10 patients showing positive tumor staining for GRP94. GRP94-positive patients showed a “non-CR” treatment response after the initial therapy or developed relapse within 1 year, resulting in a poor prognosis. In the tumor microenvironment, GRP94 expression is associated with cell survival of the TCL cells and leads to a poor prognosis. In order to overcome various stressful conditions, such as altered cellular metabolism and acidosis, TCL cells survive and lead to a poor prognosis. ( C ) PD-L1-positive patients ( n = 4, median OS, 5.5 months, p < 0.01); PD-L1-negative patients ( n = 12, median OS, 50 months, p < 0.01). PD-L1 positivity allows TCL cells to proliferate by escaping the surveillance mechanism, which results in a poor prognosis. ( D ) AKR1C3-positive patients ( n = 6, median OS, 10 months: p < 0.01); AKR1C3-negative patients ( n = 10, median OS, 62 months; p < 0.01). AKR1C3 expression in TCL cells decreases the intracellular metabolism of HO of the CHOP regimen drugs, attenuating their cytotoxic activity against the TCL cells and making the disease refractory, which results in a poor prognosis. ( E ) TP-positive patients ( n = 3, median OS, 6 months; p < 0.01); TP-negative patients ( n = 13, median OS, 56 months; p < 0.01). TP expression is mainly linked to antiapoptotic and angiogenic activities, leading to a poor prognosis. ( F ) CYP2B6-positive patients ( n = 4, median OS, 13.5 months; p < 0.05); CYP2B6-negative patients ( n = 12, median OS, 46 months; p < 0.01).

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Comparison, Immunostaining, Staining, Expressing, Activity Assay

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: HPI (TCL Urayasu classification) Group 3 (the very poor prognosis group) Case: A 33-year-old woman diagnosed with stage IIA ALK-positive ALCL (low-intermediate IPI (IPIe), PIT-Group 1). ALK positivity is generally associated with a good prognosis. However, this patient developed resistance to both CHOP and ESHAP regimens, and died after only about 2 months of treatment. The diagnosis was ALCL. Her ALCL showed positive staining for GRP94 (shown in 8) and 3 (PD-L1, TP, and GRP78, shown in 10, 12, and 20) of the other 6 poor prognostic factors (PD-L1, TP, AKR1C3, P53, PD1, and GRP78). In the tumor microenvironment, expression of PD-L1 on the cell surface blocks the immune checkpoint molecules, allowing the tumor to grow. TP is involved in starvation resistance, angiogenesis, invasion, and metastasis. GRP78 allows the tumors to overcome various stressful conditions, such as hypoxia, hypoglycemia, dysregulation of homeostasis, altered cell metabolism, and acidosis, which results in treatment resistance.

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Biomarker Discovery, Staining, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: An eNAMPT-neutralizing mAb reduces post-infarct myocardial fibrosis and left ventricular dysfunction

doi: 10.1016/j.biopha.2023.116103

Figure Lengend Snippet: Microphotographs (40X) of left ventricular (LV) sections illustrate NAMPT (A-D) and TLR4 (E-H) expression as revealed by IHC staining in both healthy and infarcted rat hearts. The nonischemic myocardium from a rat heart subjected to sham surgery (A) exhibited low, yet detectable, cytoplasmic expression of NAMPT, and an absence of TLR4 expression. IHC staining showed substantial brown precipitates of anti-NAMPT and anti-TLR4 in the peri-infarct zones of infarcted rat hearts at 6 hours (B and F), 1 week (C and G), and 4 weeks (D and H) post-IRI, indicative of the upregulation of NAMPT and TLR4.

Article Snippet: This process involved incubating the sections overnight at 4 °C with a primary rabbit anti-NAMPT pAb (Bethyl Laboratories, IHC-00011) at a 1:1000 dilution, as referenced in previous studies [ , , ].

Techniques: Expressing, Immunohistochemistry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: An eNAMPT-neutralizing mAb reduces post-infarct myocardial fibrosis and left ventricular dysfunction

doi: 10.1016/j.biopha.2023.116103

Figure Lengend Snippet: NAMPT IHC staining images of rat hearts subjected to IRI and treated with ALT-100 or saline. Panels A–B and C–D display cardiac sections (10X) illustrating Masson’s Trichrome staining (A & C) and anti-NAMPT IHC staining (B & D), respectively. Panels A & B are from a control heart treated with saline, while panels C & D are from an ALT-100-treated rat heart at 4 weeks post-IRI. The numbered boxes on the IHC images indicate areas from distinct zones selected for 40X magnification: the remote non-infarct area (“1”), the peri-infarct zone (“2”), and the fibrotic infarct region (“3”). Panels B1–B3 and D1–D3 are magnified microphotographs (40X) from the corresponding boxed areas on panels B and D, respectively. The brown staining represents NAMPT expression in the cardiac tissues. No discernible difference in the distribution patterns of NAMPT expression was qualitatively observed between the heart treated with ALT-100 and the saline control. The intensity of positive anti-NAMPT staining is lower in the cardiac section from the rat treated with ALT-100 compared to that in the saline control.

Article Snippet: This process involved incubating the sections overnight at 4 °C with a primary rabbit anti-NAMPT pAb (Bethyl Laboratories, IHC-00011) at a 1:1000 dilution, as referenced in previous studies [ , , ].

Techniques: Immunohistochemistry, Saline, Staining, Expressing

Journal: Cancer Research

Article Title: ABCB5 Maintains Melanoma-Initiating Cells through a Proinflammatory Cytokine Signaling Circuit

doi: 10.1158/0008-5472.can-14-0582

Figure Lengend Snippet: Figure 5. ABCB5 controls secretion of IL1b, an activator of IL8, to stimulate IL8. A, IL1b secretion by wild-type or DTIC-resistant A375 cells (ELISA). B, IL8 (left) or WFDC1 (right) mRNA expression by A375 WT or patient-derived melanoma cells following exogenous IL1b treatment (tr) versus control (means of n ¼ 3 independentexperiments withthree replicateseach per datapoint; , P < 0.005; , P < 0.0005). C, TLR4expression onABCB5(þ) cells(top) andABCB5()cells (bottom) in G3361 (left) and patient-derived melanoma cells (middle). Aggregate analysis for n ¼ 6 distinct melanoma specimens (G3361, A375, and SK-MEL-28 cell lines, three patient-derived specimens) is shown on the right (, P < 0.05;NS, not significant). D, immunohistochemistry staining for ABCB5 (left,brown), TLR4 (middle, blue), and costaining for ABCB5 and TLR4 (right, purple shows costaining) of a human C8161 melanoma xenograft lung metastasis (left, 400; right, 1,000 magnification). E, left, immunofluorescence costaining of clinical melanoma for TLR4 (red) and ABCB5 (green). (Continued on the following page.)

Article Snippet: Immunofluorescence staining of paraffin-embedded clinical melanoma involved TLR4 antibody (Novus Biologicals) followed by Alexa Fluor594 secondary antibody, and ABCB5 primary antibody (clone 3C2–1D12) followed by addition of biotinylated horse antimouse IgG and Alexa Fluor-488–labeled streptavidin.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Control, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Venn diagram overlapping alternatively spliced events (ASEs) genes, differentially expressed genes (DEGs), and NONO ChIP-Seq targets. B , C Co-immunoprecipitation (Co-IP) in HEK293T cells overexpressing NONO-FLAG and HOXA1-HA ( B ; n = 1), and in Day 2 hiPSC-derived differentiated cells with doxycycline-induced HOXA1-HA ( C ; n = 3), using anti-NONO and anti-HA antibodies for immunoprecipitation and detection. D , E Immunofluorescence staining showing nuclear colocalization of overexpressed HOXA1-HA and endogenous NONO in HEK293T cells ( D ) and in Day 2 differentiated cells ( E ). Specificity of the NONO antibody was validated in NONO-KO cells. Scale bar, 15 μm. n = 3. F GST pull-down assay using recombinant GST-NONO (from E. coli ) and HOXA1-HA (from HEK293T lysates). n = 2. G Schematic representation of HOXA1 and NONO protein domains: Poly-His (PH), hexapeptide (HP), homeodomain (HD), RNA recognition motifs (RRM1, RRM2), and Coiled-coil (Coil) domain. H , I Co-IP domain mapping in HEK293T cells. Interaction of NONO with HOXA1-HA truncation mutants, showing diminished binding upon HOXA1-HD deletion (ΔHD) ( H ). Interaction of HOXA1-HA with NONO-FLAG truncation mutants, showing reduced binding upon NONO-RRM2 or Coil domain deletion ( I ). n = 3. J Structural model of the NONO-HOXA1 interface, highlighting key residues (HOXA1: N230, I275, K286; NONO: E278, Q281, R287). K , L Co-IP validation using point mutants in HEK293T cells. Results show disrupted interaction with the HOXA1-K286A (NONO) mutant ( K ) and the NONO-E278A (HOXA1) mutant ( L ) n = 3. M , N Cycloheximide (CHX) chase assay to assess HOXA1-HA stability. Quantification of HOXA1-HA in nuclear lysates of WT and NONO-KO cells ( M ). Quantification of HOXA1-HA in HEK293T cells co-expressing WT or E278A NONO-FLAG ( N ). Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. Data are presented as mean values ± SD. n represents independent biological replicates. Source data are provided as a Source Data file.

Article Snippet: For colocalization studies of NONO and HOXA1, staining involved antibodies against NONO (Santa Cruz, sc-376865) at a 1:200 dilution and HA tag (Abcam, ab9110) at a 1:500 dilution.

Techniques: ChIP-sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Immunofluorescence, Staining, Pull Down Assay, Recombinant, Binding Assay, Biomarker Discovery, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Genome browser visualization of ChIP-seq tracks at key cardiac gene loci. B GO biological process enrichment analysis of selected terms for genes bound by NONO in Day 2 differentiated Cells. C Venn diagram of NONO-enriched and HOXA1-HA enriched overlap peaks in Day 2 differentiated cells. D GO biological process enrichment analysis of selected terms for genes of NONO and HOXA1-HA co-binding. E Heatmaps of HOXA1 ChIP-seq signals in WT and NONO-KO cells. HOXA1 binding is significantly reduced in the absence of NONO, with shared and lost peaks shown. F Representative ChIP-seq tracks of HOXA1 binding at precardiac mesoderm loci ( MESP1 and PDGFRA ) in Day 2 WT and NONO-KO cells. G ChIP-qPCR analysis showing reduced HOXA1 binding at target loci in Day 2 differentiated NONO-KO cells compared to WT. n = 3. H GO enrichment analysis of genes with lost HOXA1 binding in NONO-KO cells and differential expression (DEGs) in HOXA1-KO cells. I qRT-PCR analysis of HOXA1-regulated genes in Day 2 WT, NONO-KO, and HOXA1-KO cells. n = 3. J Co-immunoprecipitation (Co-IP) assessment of NONO homodimerization in Day 2 cells expressing Dox-induced NONO-FLAG and NONO-HA. n = 3. K , L Co-IP analysis of HOXA1 homodimerization in Day 2 WT and NONO-KO cells co-expressing HOXA1-HA and HOXA1-FLAG, using anti-HA ( K ) and anti-FLAG ( L ) antibodies for immunoprecipitation, followed by immunoblotting with anti-FLAG and anti-HA antibodies. Note the impaired HOXA1 self-association in NONO-KO cells. n = 3. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT-positive cardiomyocytes in NONO-KO and NONO/HOXA1 Double-KO (DKO) at Day 15. n = 3. O , P Immunostaining ( O ) and quantification ( P ) of sarcomere organization (α-ACTININ, green; cTNT, red) in Day 15 NONO-KO and DKO cardiomyocytes. Scale bar, 15 μm. ( n = 4; NONO-KO, 27 cells; DKO, 30 cells). Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test, except for ( P ) (Fisher’s exact test). P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Article Snippet: For colocalization studies of NONO and HOXA1, staining involved antibodies against NONO (Santa Cruz, sc-376865) at a 1:200 dilution and HA tag (Abcam, ab9110) at a 1:500 dilution.

Techniques: ChIP-sequencing, Binding Assay, ChIP-qPCR, Quantitative Proteomics, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot, Flow Cytometry, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Heatmap showing log2-fold changes of differentially expressed genes (DEGs) among NONO-dependent and HOXA1-regulated WNT pathway genes in NONO-KO (left) and HOXA1-KO (right) cells. B Representative ChIP-seq tracks of WNT pathway genes showing HOXA1 binding in Day 2 WT cells, with reduced binding in NONO-KO cells. C , D Western blot analysis of cytoplasmic and nuclear β-CATENIN levels in Day 2 differentiated cells. WT vs. HOXA1-KO ( C ). WT, NONO-KO, and NONO-RE ( D ). n = 3. E , F Cycloheximide (CHX) chase assays and quantification of nuclear β-catenin stability in NONO-KO ( E ) and HOXA1-KO ( F ) cells. Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. G – J TOP/FOP reporter assays in HEK293T cells treated with or without CHIR99021 (CHIR). Dose-dependent knockdown of HOXA1 ( G ) (siHOXA1; n = 5). Dose-dependent overexpression of HOXA1 ( H ) ( n = 5). Dose-dependent knockdown of NONO ( I ) (siNONO; n = 6). Dose-dependent overexpression of NONO ( J ) ( n = 5). K TOP/FOP reporter assay in cells overexpressing full-length HOXA1 or homeodomain-deleted mutant (HOXA1 ΔHD), treated with or without CHIR. n = 4. L TOP/FOP reporter assay in cells expressing WT or K286A mutants (MU) HOXA1, followed by NONO overexpression. Cells were treated with or without CHIR. n = 5. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT + cardiomyocytes in Day 15 NONO-KO cultures treated with CHIR (8–14 μM) during Days 0–2. n = 3. O qRT-PCR analysis of TNNT2 , MYH6 , and MYH7 expression in Day 15 NONO-KO cardiomyocytes treated with CHIR (8–14 μM) during Days 0–2. n = 3. P Schematic model of the NONO-HOXA1-Wnt signaling axis in cardiomyocyte differentiation. Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test. P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Article Snippet: For colocalization studies of NONO and HOXA1, staining involved antibodies against NONO (Santa Cruz, sc-376865) at a 1:200 dilution and HA tag (Abcam, ab9110) at a 1:500 dilution.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Knockdown, Over Expression, Reporter Assay, Mutagenesis, Expressing, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: DHX9 is essential for induction of IL6 and type I IFN upon MHV-68 infection. (A–D) shDHX9/3T3 ( A, B ) and shDHX9/HeLa ( C, D ) cells were mock-infected or infected with MHV-68 at MOI 2 for 6 and 12 h. Total cellular RNA was extracted and subjected to RT-qPCR for IL6 (A, C) and IFN-β (B, D) mRNA levels. ( E, F ) shDHX9/HeLa and shCtrl cells were transfected with 2 × кB-luc ( E ) or IFN-β-luc ( F ) reporter plasmid (100 ng) for 20 h, then infected with MHV-68 at MOI 2 for 12 h, and harvested for luciferase assays. ( G ) shDHX9/HeLa and shCtrl cells were transfected with 2 × кB-luc (100 ng) and HA-p65 (50 ng) for 28 h and harvested for luciferase assays. The relative luciferase activity (RLA) of each sample was determined by normalizing the activity to the sample's β-galactosidase activity. The data represent the mean ± SD of triplicate assays. *** P < 0.001 versus control, ns = nonsignificant (Student's t test).

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: Endogenous DHX9 interacts with NF-κB p65 in MHV-68–infected fibroblasts and epithelial cells. ( A ) HEK293T cells were transfected with FLAG-DHX9 (3 μg) and HA-p65 (3 μg) for 36 h and infected with MHV-68 at MOI 2 for 12 h. Cell lysates were subjected to immunoprecipitation (IP) with an anti-FLAG antibody and western blot analysis with an anti-HA antibody. ( B ) Subcellular fractions of mock-infected and MHV-68-infected HeLa cells at MOI 2 were prepared at the indicated time points. PARP-1 and α-tubulin were used to confirm the integrity of nuclear and cytosolic extracts, respectively. ( C ) Two representative confocal microscopy images of HeLa cells that were either mock-infected or infected with MHV-68 at MOI 2 for 12 h. The cells were fixed and stained for endogenous DHX9 (FITC-green) and p65 (Cy3-red). DAPI staining (blue) was used to visualize nuclei. (D and E) Interactions of DHX9 with NF-κB p65 and RNAPII in fibroblasts and epithelial cells during viral infection. NIH3T3 cells ( D ) and HeLa cells ( E ) were mock-infected or infected with MHV-68 at MOI 2 for 12 h. The cytosolic and the nuclear extracts from the cells were subjected to immunoprecipitation with the anti-DHX9 antibody and immunoblotted with the indicated antibodies. The input lysates represent 10% of the extracts by volume used for each immunoprecipitation. ( F ) The cells were prepared as described in (C) and stained for endogenous DHX9 (FITC-green) and RNAPII (Cy3-red). All panels show ×1000 magnification (scale bar: 20 μm).

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: Endogenous DHX9 is required for RNAPII recruitment to NF-κB–dependent antiviral promoters. ( A ) shCtrl and shDHX9/HeLa cells were infected with MHV-68 at MOI 2 for 12 h. The nuclear extract was subjected to immunoprecipitation with the anti-p65 antibody and immunoblotted with the indicated antibodies. The input lysates represent 10% of nuclear extracts by volume used for each immunoprecipitation. (B–D) shCtrl/HeLa and shDHX9/HeLa cells were infected with MHV-68 at MOI 2 for 12 h. Cell lysates were prepared and analyzed by a ChIP assay with an anti-p65 or anti-RNAPII antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of precipitated DNAs were measured by qPCR using primers flanking the promoter regions of IL6 ( B ), IFN-β ( C ), and GAPDH ( D ). The data represent mean ± SD of triplicate assays.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Immunoprecipitation, Control

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: The ATPase/helicase activity of DHX9 is required for RNAPII recruitment to NF-κB–dependent antiviral promoters. Stable cells expressing 3′ UTR shDHX9 were reconstituted with FLAG-DHX9-WT (lacking the 3′ UTR), K417R, ΔDUF, ΔDUF+NTD DHX9 , or ΔDUF+NLS SV40 (18 μg) for 36 h. The transfected cells were either mock-infected or infected with MHV-68 at MOI 2 for 12 h. ( A ) Cell lysates were subjected to western blot analysis with anti-DHX9 and anti-α-tubulin antibodies to assess the expression levels. (B–D) Cell lysates were analyzed by ChIP assays involving the anti-p65 or anti-RNAPII antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of input and precipitated DNAs were measured by qPCR with primers flanking the promoter regions of IL6 ( B ), IFN-β ( C ), and ‘ GAPDH ( D ). The data represent mean ± SD of triplicate assays.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Activity Assay, Expressing, Transfection, Infection, Western Blot, Control, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: DHX9 is recruited to the NF-κB p65–dependent promoters during viral infection. HeLa cells were either mock-infected or infected with MHV-68 at MOI of 2 for 12 h. (A and B) Cell lysates were prepared and analyzed by ChIP assays with an anti-p65, anti-RNAPII, or anti-DHX9 antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of precipitated DNA were measured by quantitative PCR (qPCR) involving primers flanking the promoter regions of IL6 ( A ) and IFN-β ( B ). The data represent mean ± SD of triplicate assays. ( C ) The cell lysates were prepared for ChIP, first subjected to immunoprecipitation with the anti-p65 antibody and then subjected to re-ChIP with anti-RNAPII and anti-DHX9 antibodies. Genomic DNA samples sequentially enriched in the NF-κB target gene promoters ( IL6 and IFN-β promoters) were amplified by PCR and analyzed by agarose gel electrophoresis (lanes 3–6). Reciprocal re-ChIP using the anti-RNAPII or anti-DHX9 antibody was also conducted with the first antibody followed by incubation with the second antibody as indicated (lanes 7–14). Normal IgG was included as a control for nonspecific immunoprecipitation and input lanes indicate the chromatin complexes prior to immunoprecipitation. A set of representative agarose gel images is shown from two independent experiments.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Incubation

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: Efficacy evaluation of adoptive T-cell therapy in C57BL/6 mice. a Cell count from inguinal lymph node (left) and spleen (right) of cyclophosphamide (CTX)-treated C57BL/6 mice. Mice were intraperitoneally injected with the indicated doses (mg/kg) of CTX. n = 3 mice per each point. Error bars indicate means ± SD. b Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; treated ex vivo-primed Thy1.1 + Pmel-1 CD8 + T (ex-T) cells, 2 × 10 6 /mouse. Two days prior to ex-T-cell transfer, mice were pre-conditioned with 300 mg/kg CTX. Mice with anti-CD4 post conditioning were treated every week with anti-CD4 antibody from day 10 for 5 weeks. c Tumor growth curves are shown. Each curve indicates longitudinal changes of an individual mouse. † indicates the death of an individual mouse at the indicated time point. The number of mice surviving up to day 80 is shown. d Survival rate per group is indicated; two-tailed log-rank (Mantel–Cox) tests were used to determine statistical significance. e Representative images of mice with and without vitiligo. f Number of mice with vitiligo and having no sign of tumor on day 80. c – f n = 10 mice/group. αCD4, anti-CD4 antibody. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Cell Counting, Injection, Ex Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: a Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex vivo-primed Thy1.1 + Pmel-1 CD8 + T (ex-T) cells, 2 × 10 6 /mouse. b – d Marker expression and subset analysis of ex-T and endogenous CD8 + T (en-T) cells. c The proportion of naive (CD62L + CD44 − ), EM (effector memory; CD62L − CD44 + ), and CM (central memory; CD62L + CD44 + ) subsets were calculated. e – j Analysis of the proliferation of ex vivo-primed Thy1.1 Pmel-1 CD8 + T (Thy1.1 + ) cells and unstimulated polyclonal CD8 + T (CD45.1 + ) cells in the presence or absence of tumor antigen. e Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; Thy1.1 + , 1 × 10 6 /mouse; CD45.1 + , 1 × 10 6 /mouse. f The cell count of Thy1.1 + and CD45.1 + cells. g Fold change in cell number over the average of the non-tumor CTX pre group. h Representative flow cytometry images of Thy1.1 + and CD45.1 + cells analyzed for CD44 expression. The number of cells with more than seven divisions ( i ) and of CD44 expression ( j ) were calculated. n = 4 mice per group. Each symbol indicates the calculated value of an individual mouse. Horizontal bars indicate means. Two-tailed unpaired Student’s t -test. CD4 post , anti-CD4 post conditioning; CFSE, carboxyfluorescein succinimidyl ester; CTV, Celltrace Violet; CTX pre , cyclophosphamide preconditioning; PBS, phosphate-buffered saline. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Injection, Ex Vivo, Marker, Expressing, Cell Counting, Flow Cytometry, Two Tailed Test, Saline

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: a Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex vivo-primed Thy1.1 + Pmel-1 CD8 + T (ex-T) cells, 2 × 10 6 /mouse. b Analysis of polyfunctionality. Cells were stimulated and analyzed for cytokine expression as in Supplementary Fig. . SPICE v6.0 was used to draw the pie chart. Arc length indicates the proportion of cells expressing the corresponding marker. Pie area indicates the proportion of cells expressing the given number of functional markers. n = 5 mice per group. c CD25 expression was analyzed in lymphocytes incubated with (activated) or without (basal) B16-F10 for 16 h. d The killing efficiency of endogenous CD8 + (en-T) cells against B16-F10 was evaluated. Cells from four mice of each group were pooled and assayed in biological triplicates. Error bars indicate means ± SEM. Two-way ANOVA with Bonferroni post hoc test was used to determine statistical significance. e Shannon evenness (normalized Shannon–Wiener index) of TCR clonotype analysis. TCR α-chain (left) and TCR β-chain (right) of en-T cells were analyzed. CTX pre , n = 3 mice; CTX pre /CD4 post , n = 5 mice. Error bars indicate means ± SD. d , e En-T cells isolated as in Supplementary Fig. were used. f – i Characterization of tumor-infiltrating CD8 + T cells. f Schematic of the tumor tissue experiments. Injected cell number: B16-F10, 2 × 10 6 /mouse; ex-T cells, 2 × 10 6 /mouse. g The count of ex-T and en-T cells among 100 live cells from tumor tissues were calculated using flow cytometry data. h The proportion of naive (CD62L + CD44 − ), EM (effector memory; CD62L − CD44 + ), and CM (central memory; CD62L + CD44 + ) subsets were calculated. i The frequency of cells expressing PD-1 in ex-T and en-T cells are shown. c , g – i n = 4 mice per group. c , e , g – i Each symbol indicates the calculated value of an individual mouse. Horizontal bars indicate means. c , g – i Two-tailed unpaired Student’s t -test. CD4 post , anti-CD4 post conditioning; CTX pre , cyclophosphamide preconditioning. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Injection, Ex Vivo, Expressing, Marker, Functional Assay, Incubation, Isolation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: a Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex vivo-primed Thy1.1 + Pmel-1 CD8 + T (ex-T) cells, 2 × 10 6 /mouse. b Differentially expressed genes in microarray analysis. Genes that were upregulated (>3-fold) in CTX pre /CD4 post compared with that in CTX pre -experienced endogenous CD8 + T (en-T) cells are indicated. En-T cells isolated as in Supplementary Fig. were used. For each group, en-T cells from five mice were pooled and used for analysis. c IL-18Rα expression in en-T cells was analyzed. n = 5 mice per group. Two-tailed unpaired Student’s t -test. d – f Analysis of TCR distribution and gene expression profiles of IL-18Rα hi en-T cells. d Schematic of analyte preparation. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex-T cells, 2 × 10 6 /mouse. On day 25, IL-18Rα hi -enriched en-T cells were isolated via magnetic cell separation. e Shannon evenness (normalized Shannon–Wiener index) of TCR α-chain (left) and β-chain (right) are shown. n = 3 mice per group. f Whole transcriptome analysis. Genes that were upregulated (>3-fold) in IL-18Rα hi -enriched en-T cells compared with en-T cells in the flow-through are indicated. n = 4 mice per group. g Multi-color flow cytometry analysis of en-T cells. Analytes prepared as in a were stained with the indicated markers and analyzed. Dimensionality reduction (t-SNE; t -distributed stochastic neighbor embedding) was performed for visualization. c , e Each symbol indicates an individual mouse. Horizontal bars indicate means. xCD4 post , anti-CD4 post conditioning; CTX pre , cyclophosphamide preconditioning. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Injection, Ex Vivo, Microarray, Isolation, Expressing, Two Tailed Test, Gene Expression, Magnetic Cell Separation, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: a Polyfunctionality of IL-18Rα low , IL-18Rα mid , and IL-18Rα hi endogenous CD8 + T (en-T) cells was calculated as in Fig. using the data from Supplementary Fig. . Analytes prepared as in Fig. were used. b , c Evaluation of anti-tumor efficacy of IL-18Rα hi en-T cells. b Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex vivo-primed Thy1.1 + Pmel-1 CD8 + T (ex-T) cells, 2 × 10 6 /mouse; isolated en-T cells (from the 1st transfer mice), 1 × 10 6 /mouse. c Tumor growth until day 32 is shown. Tumor growth curves over the whole period and survival curves are shown in Supplementary Fig. . IL-18Rα hi en-T indicates magnetically enriched cells as shown in Supplementary Fig. . Error bars indicate means ± SEM. d IL-18Rα expression in ex-T cells was analyzed as in Fig. . Each symbol indicates the calculated value of an individual mouse. Horizontal bars indicate means. e Polyfunctionality of IL-18Rα low , IL-18Rα mid , and IL-18Rα hi ex-T cells was calculated as in Fig. using the data analyzed in Supplementary Fig. . d , e Analytes prepared as in Fig. were used. f Cytokine concentration in culture media was evaluated. Isolated IL-18Rα low/mid and IL-18Rα hi ex-T cells as in Supplementary Fig. were co-cultured with gp100 25–33 -pulsed or unpulsed C57BL/6 splenocytes for 4 h. IL-18 (30 ng/mL) was added to the treatment group. Pooled cells from CTX pre /CD4 post -experienced mice ( n = 5) were used in this assay. Biological triplicates. g , h Evaluation of anti-tumor efficacy of IL-18Rα hi ex-T cells. g Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex-T cells, 2 × 10 6 /mouse; isolated ex-T cells (from the 1st transfer mice), 5 × 10 5 /mouse. h Tumor growth until day 32 is shown. Tumor growth curves over the whole period and survival curves are shown in Supplementary Fig. . IL-18Rα hi and IL-18Rα low/mid ex-T mean magnetically sorted cells as in Supplementary Fig. . Error bars indicate means ± SD. i – k Evaluation of the role of IL-18 signal in CTX pre /CD4 post regimen. i Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex-T cells, 2 × 10 6 /mouse. Tumor growth ( j ) and survival rates ( k ) are shown. j Each curve indicates an individual mouse. † indicates death at the indicated time point. The number of mice surviving up to day 80 is represented. g – k Anti-IL-18 treatment started from day 4 and was performed thrice a week for the whole period. a – e, i – k n = 5 mice/group. g , h WT groups, n = 7 mice/group; Il18r1 knockout groups, n = 5 mice/group. Two-tailed unpaired Student’s t -test ( d ) and two-tailed log-rank (Mantel–Cox) tests ( k ) were used to determine statistical significance. αIL-18, IL-18 neutralizing antibody; αMHC-1, anti-MHC-1 blocking antibody; CD4 post , anti-CD4 post conditioning; CTX pre , cyclophosphamide preconditioning; WT, wild-type mice. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Injection, Ex Vivo, Isolation, Expressing, Concentration Assay, Cell Culture, Knock-Out, Two Tailed Test, Blocking Assay

Journal: Nature Communications

Article Title: Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18Rα hi CD8 + T cells

doi: 10.1038/s41467-021-25559-7

Figure Lengend Snippet: a – c Evaluation of variables that led to IL-18Rα hi cell generation. The treatment with full regimen (as in Fig. ) or a modified regimen that lacks each variable was applied to mice ( n = 5 mice per group). On day 25, the ratio ( a ) and the number ( b ) of IL-18Rα hi Thy1.1 + Pmel-1 CD8 + T (ex-T) cells in lymphoid tissues were analyzed. c The correlation between lymphopenia score (represented as −log [total cell count in spleen and tumor-draining lymph nodes]) and the proportion of IL-18Rα hi ex-T cells was evaluated. d – f Visualization of single-cell transcriptome analysis of CTX pre and CTX pre /CD4 post -experienced ex-T cells. Cells from four mice of each group were pooled and analyzed. d After unsupervised clustering, the ex-T cells were projected on two-dimensional space using t -distributed stochastic neighbor embedding (t-SNE). The information related to groups, clusters, and Il18r1 expression are shown. e Genes that are differentially expressed compared with those in the cluster 6 are listed. f The log-normalized transcript levels of the indicated genes are shown. g , h Evaluation of the effect of IL-7 signal on IL-18Rα hi CD8 + T-cell generation. g Schematic of the experiment. Injected cell number: B16-F10, 2 × 10 5 /mouse; ex-T cells, 2 × 10 6 /mouse. The treatment group received daily treatment of non-lytic Fc-fused IL-7 (IL-7-Fc) from day 10–14. h Representative data, the ratio, and the number of IL-18Rα hi ex-T cells are shown. a , b , h n = 5 mice per group. Each symbol indicates the calculated value of an individual mouse. Horizontal bars indicate means. * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA with the Dunnett’s post hoc test ( a , b ; the first group was the control), two-tailed Spearman’s rank correlation ( c ), and two-tailed unpaired Student’s t -test ( h ) were used to determine statistical significance. αIL-18, IL-18 neutralizing antibody; CD4 post , anti-CD4 post conditioning; CTX pre , cyclophosphamide preconditioning; DT, diphtheria toxin; WT, wild-type mice. Source data are provided as a Source Data file.

Article Snippet: Starting from day 10, anti-CD4 post conditioning involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5; BioXCell, NH, USA) for up to 5 weeks.

Techniques: Modification, Cell Counting, Expressing, Injection, Control, Two Tailed Test