Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in cell lines. ( A ) mRNA normalized TPM of IL4Rα from Human Protein Atlas for Daudi and SiHa cell lines. ( B ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL4-Bevacizumab in Daudi and SiHa cell lines. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. Means ± SEM of three biological replicates are shown. ( C ) mRNA normalized TPM of IL7R from Human protein atlas for Daudi and SiHa cell lines. ( D ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL7-Bevacizumab in Daudi and SiHa cell lines. Curves are best fits for three-parameter nonlinear regressions. Mean values ± SEM are from three biological replicates. ( E ) Diagram of coculture experiments. Daudi cells (gray) and SiHa cells (purple) are coincubated with VEGF-647 and either IL4-Bevacizumab kineTAC (green) or IL7-Bevacizumab kineTAC (blue). Cell type–specific receptor expression allows for cell-specific internalization of VEGF using the respective kineTAC. ( F ) Representative flow cytometry data from coculture experiment. Daudi and SiHa cells in equal amounts were incubated for 24 h with 25 nM VEGF-647, and 0.3 nM IL4-Bevacizumab or 10 nM IL7-Bevacizumab. Gates show thresholds for VEGF positivity, defined as approximately 1% of total cells for VEGF-647 only. Top row: FITC + Daudi GFP . Bottom row: FITC − SiHa. Percentages in gate are displayed. ( G ) Fold change in VEGF-647 median fluorescence intensity in the coculture Daudi and SiHa experiment from F . Mean fold change over 25 nM VEGF-647 alone ± SEM is presented. The asterisk represents a discovery (q < 1%) using the false discovery rate to correct for multiple comparisons. ( H ) Coculture VEGF-647 percent positivity (same gate as in F ) as a dose response of IL4-Bevacizumab. Mean values and ± SEM are from three biological replicates. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. ( I ) same as in H , but for IL7-Bevacizumab.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in primary lymphocytes and PBMCs. ( A ) Diagram depicting tissue-specific expression of potential kineTAC receptors. ( B ) tSNE analysis of IL4Rα expression from scRNA seq of two PBMC donors, obtained from the Immune Cell Atlas ( 57 ). Notice the prominent naïve B cell cluster of high IL4Rα expressors (green). ( C ) tSNE analysis of IL7R expression from the same dataset. Notice primarily T cell restricted expression of IL7R (blue). ( D ) Immune Cell Atlas cluster annotations for primary cell lineages, colored by lineage as in legend to the right. ( E ) Raw VEGF-647 median fluorescence intensities across two donors for isolated CD19+ cells from four technical replicates. Cells were incubated with 25 nM VEGF-647 and 10 nM of indicated kineTAC or isotype. ( F ) same as E , but using CD3+ T-cells. ( G and H ) VEGF-647 with PBMC components. 10 nM of the indicated kineTAC or isotype was added to 25 nM VEGF-647 along with 200 k cells/well PBMCs and allowed to internalize for 24 h. Percent VEGF positive was defined by the no VEGF-647 condition ( SI Appendix , Fig. S6 ). Analyzed by repeated measures donor-paired one-way ANOVA, with P values for Dunnett’s correction for multiple comparisons to the kineTAC of interest (IL4-Beva for B cells, IL7-Beva for T cells) (* P < 0.05, ** P < 0.01). ( G ) Analysis of B cell gate and h analysis of T cell gate.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Fluorescence, Isolation, Incubation

Journal: Frontiers in Immunology

Article Title: Ex vivo- generated lymphoid progenitors encompass both T cell and innate lymphoid cell fates

doi: 10.3389/fimmu.2025.1617707

Figure Lengend Snippet: Production of ProTcell batches and their characterization at single cell level. (A) Experimental scheme of the ProTcell culture system: CD34 + cells, originated from either CB or mPB, are seeded in a culture unit coated with Retronectin and DLL4-Fc with a cocktail of cytokines in αMAM. After 7 days of culture, T cell progenitors are harvested and analyzed by flow or mass cytometry and single cell RNA sequencing. (B) Contour plot of flow cytometry analysis depicting the expression of CD7 and CD34 in a representative CB (right) or mPB (left) ProTcell product. (C–H) Further transcriptomic analyses were focused on cells positive for the CD7 transcript. (C) scRNAseq data was visualized by UMAP dimension reduction technique and 7 cell clusters were identified (Resolution 0.6). (D) Violin plots showing the normalized expression level of T lymphoid-associated genes in the 7 clusters. Density plots illustrating the gene expression of (E) T cell identity genes GATA3, TCF7, BCL11b and CD7, and (F) markers of developing T lymphoid IL7R, CD3D, CD3E and CD3G. (G) Clustered dot plot depicting gene expression of several markers in the 7 ProTcell clusters. Dot size indicates the percentage of cells expressing marker-specific genes in each cluster. Average expression levels of cluster-specific genes are depicted according to the color scale shown (blue represents low; red represents high). (H) Heatmaps illustrating the similarity scores between each individual cell from ProTcell data and annotated clusters from Cordes et al. (upper panel) and Lavaert et al. (lower panel) data. Scale bar: Z scores of the relative Spearman coefficients. Each column is representative of a single ProTcell scored across each population indicated by the row name.

Article Snippet: 165Ho , CD127 (IL7R) , Fluidigm , A019D5 , 3165008C.

Techniques: Mass Cytometry, RNA Sequencing, Flow Cytometry, Expressing, Gene Expression, Marker

Journal: eLife

Article Title: Reprogramming of GM-CSF-dependent alveolar macrophages through GSK3 activity modulation

doi: 10.7554/eLife.102659

Figure Lengend Snippet: ( A ) DoRoThEA analysis on the ranked comparison of the DMSO-GM-MØ and CHIR-GM-MØ transcriptomes. ( B ) Gene set enrichment analysis (GSEA) of MAFB-regulated gene sets (from GSE155719 ) on the ranked comparison of the DMSO-GM-MØ and CHIR-GM-MØ transcriptomes. NES and FDRq values are indicated in each case. ( C ) Overlap between the genes upregulated (|log2FC|>1; adjp<0.05) in CHIR-GM-MØ (relative to DMSO M-MØ) and MAFB-dependent genes (from GSE155719 , left panel) or the 75-geneset of MAFB-regulated genes (right panel) , with indication of some of the overlapping genes. ( D ) Relative mRNA levels of the indicated genes in DMSO-GM-MØ and CHIR-GM-MØ, as determined by RNA-Seq on three independent samples ( GSE256208 ). Adjp of the comparison, shown in each case, was assessed using DESeq2. ( E ) CD163, MAF, and IL7R protein levels in DMSO-GM-MØ and CHIR-GM-MØ, as determined by western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the MAF/GAPDH, IL7R/GAPDH, and CD163/GAPDH protein ratios from seven independent experiments are shown (right panels) (paired Student’s t-test: ***, p<0.005; ****, p<0.001). A representative western blot experiment is shown in each case. ( F ) FOLR2 protein levels in DMSO-GM-MØ and CHIR-GM-MØ, as determined by western blot (left panel). Vinculin protein levels were determined as protein loading control. Mean ± SEM of the FOLR2/Vinculin protein ratio from three independent experiments is shown (lower panel) (paired Student’s t-test: **, p<0.01). A representative western blot experiment is shown. ( G ) Production of the indicated soluble factors by DMSO-GM-MØ and CHIR-GM-MØ, as determined by ELISA. Mean ± SEM of seven independent samples are shown (paired Student’s t-test: *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001). Figure 2—source data 1. Discriminant regulon expression analysis (DoRothEA) of DMSO-GM-MØ and CHIR-GM-MØ transcriptomes. Top 15 transcription factors are shown. Figure 2—source data 2. mRNA levels (read counts) of the indicated genes in DMSO-GM-MØ and CHIR-GM-MØ, as determined by RNA-Seq on three independent experiments. Figure 2—source data 3. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 4. Original files for western blot analysis displayed in . Figure 2—source data 5. Protein ratios of MAF/GAPDH, IL7R/GAPDH, and CD163/GAPDH in DMSO-GM-MØ and CHIR-GM-MØ from seven independent experiments and statistical analysis. Figure 2—source data 6. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 7. Original files for western blot analysis displayed in . Figure 2—source data 8. Protein ratios of FOLR2/Vinculin in DMSO-GM-MØ and CHIR-GM-MØ from three independent experiments and statistical analysis. Figure 2—source data 9. Concentration of CCL2, CCL18, IL10, LGMN, SPP1, and Activin A in DMSO-GM-MØ and CHIR-GM-MØ from seven independent experiments and statistical analysis.

Article Snippet: After blocking the unoccupied sites with 5% non-fat milk diluted in Tris-Buffered Saline plus Tween 20 (TBS-T), protein detection was carried out with antibodies against MAFB (HPA005653, Sigma-Aldrich), total GSK3α (#4337, Cell Signaling Technology), total GSK3β (#27C10, Cell Signaling Technology), total GSK3 (#5676, Cell Signaling Technology), p-Ser 9 -GSK3β (#5558, Cell Signaling Technology), p-Ser 21 -GSK3α (#9316, Cell Signaling Technology), p-Tyr 279 /P-Tyr 216 -GSK3α/β (#05-413, Sigma-Aldrich), CD163 (#MCA1853, Bio-Rad), MAF (sc-518062, Santa Cruz Biotechnology), IL7R (sc-514445, Santa Cruz Biotechnology) and FOLR2 (clone 94b, kindly provided by Dr. Takami Matsuyama) , and Vinculin (#V9131, Sigma-Aldrich) or GAPDH (sc-32233, Santa Cruz Biotechnology) as protein loading controls.

Techniques: Comparison, RNA Sequencing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay