Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC binds HSPA8 to induce ferroptosis in HCC. (A) Schematic diagram of the drug Affinity responsive Target stability (DARTS) assay workflow. (B) Representative coomassie blue-stained SDS-PAGE gel of DARTS samples. (C) Bubble plot summarizing MS results: x-axis shows δsequence coverage (GAC vs. control), bubble size represents protein score. (D) Protein-protein interaction (PPI) network of DARTS-identified targets; nodes are colored by DMNC algorithm score (red = high centrality, yellow = low). (E–F) Cellular thermal shift assay (CETSA) confirms GAC binding stabilizes HSPA8. Representative immunoblots (E) of HSPA8 levels in lysates from HCCLM3 cells treated with GAC or DMSO and subjected to a temperature gradient (37-65 °C) and quantification (F) of relative HSPA8 levels at each temperature, data were assessed by the repeated-measures ANOVA, n = 3 in each group. (G–H) Microscale thermophoresis (MST): representative MST traces (G); dose-response curve showing GAC binding to recombinant HSPA8 (H). (I–J) molecular docking pose(I) of GAC bound to HSPA8 and magnified view of the molecular docking pose (J) focusing on the interactions between GAC and key residues (Tyr15, Gly201, Arg272, Asp366). (K–M) molecular dynamics (MD) simulations of GAC bound to HSPA8: RMSD of HSPA8 backbone over 100 ns (K); RMSF per residue(L); per-residue energy decomposition identifying key interacting residues (M). (N) Schematic of HSPA8 point mutations generated at predicted GAC-binding residues. (O-P) CETSA validation of HSPA8 mutants: Western blots (O) and quantification (P) demonstrate loss of thermal stabilization by GAC in HSPA8 MUT , n = 3 in each group. (Q–R) Flow cytometry histogram of Bodipy665/676 intensity and quantification of the percentage of oxidized cells in shHSPA8 vs. scramble cells treated with GAC or not, n = 3 in each group. (S–T) Knock down HSPA8 restores redox homeostasis: Quantified MDA levels, data were assessed by the Mann-Whitney test. (S) and GSH/GSSG ratio (T), n = 3 in each group. (U-V) Flow cytometry plots (U) and quantification (V) of the percentage of Annexin V + cells in shHSPA8 vs. scramble cells treated with GAC or not, n = 3 in each group. (W-X) Knock down HSPA8 ameliorates clonogenic capacity: Representative colony formation images(W) and quantified colony counts (X), n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Staining, SDS Page, Control, Thermal Shift Assay, Binding Assay, Western Blot, Microscale Thermophoresis, Recombinant, Residue, Generated, Biomarker Discovery, Flow Cytometry, Knockdown, MANN-WHITNEY

Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC promotes CMA-dependent GPX4 degradation to induce ferroptosis. (A–B) GAC induces HSPA8-dependent KFERQ puncta formation, indicating CMA activation. Representative images of confocal images (A) detecting the KFERQ-probe (bar = 25 μm) and quantification of KFERQ puncta per cell (B) using ImageJ, n = 3 in each group. (C) CCK-8 assay assessing cytotoxicity in cells pretreated with indicated concentrations of Bafilomycin A1 (BafA1) for 12 h before GAC treatment, n = 3 in each group. (D–E) Flow cytometry histogram of Bodipy-C11 intensity (D) and quantification (E) of the percentage of oxidized cells in HCCLM3 cells pretreated with BafA1 before GAC treatment, n = 3 in each group. (F–G) Flow cytometry plots (F) and quantification (G) of the percentage of Annexin V + cells in HCCLM3 cells pretreated with BafA1 before GAC treatment, n = 3 in each group. (H) Table generated by KFERQ-finder showing the gene names, KFERQ-like motifs, and their starting positions within GPX4 and SLC7A11. (I-K) HSPA8 knockdown specifically prevents GAC-induced GPX4 degradation. Representative immunoblots (I) and quantification of GPX4 (J) and SLC7A11(K), normalized to β-actin, n = 3 in each group. (L–N) LAMP2A knockdown abolishes GAC-induced GPX4 degradation. Representative immunoblots (L) and quantification of LAMP2A (M) and GPX4 (N), normalized to β-actin, n = 3 in each group. (O–P) Flow cytometry histogram of Bodipy-C11 intensity (O) and quantification (P) of the percentage of oxidized cells in shLAMP2A vs. scramble cells treated with GAC or not, n = 3 in each group. (Q-R) Flow cytometry plots (Q) and quantification (R) of the percentage of Annexin V + cells in shLAMP2A vs. scramble cells treated with GAC or not, n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Activation Assay, CCK-8 Assay, Flow Cytometry, Generated, Knockdown, Western Blot

Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC binds HSPA8 to promote HSPA8-GPX4 interaction via KFERQ-like motif. (A) GAC enhances the endogenous HSPA8-GPX4 interaction. Co-immunoprecipitation (co-IP) of GPX4 followed by immunoblotting for HSPA8 in HCCLM3 (left panel) and HepG2 (right panel) cells, n = 3 in each group. (B-D) GAC increases HSPA8-GPX4 co-localization. Representative immunofluorescence confocal microscopy images (B) showing endogenous GPX4 (red) and HSPA8 (green). Nuclei were stained with DAPI (blue). Scale bar: 25 μm. Line-scan intensity profiles (C) across representative regions of interest (ROIs) from (B), demonstrating correlated fluorescence signals of HSPA8 and GPX4 at subcellular resolution. Quantification (D) of co-localization using Pearson’s correlation coefficient (analyzed with the JACoP plugin in ImageJ), n = 3 in each group. (E) Co-IP of GPX4 in cells expressing wild-type HSPA8 (HSPA8 WT ) or binding-deficient mutant HSPA8 (HSPA8 MUT ); GAC enhances HSPA8-GPX4 interaction only in the presence of HSPA8 WT .(F) schematic of GPX4 highlighting two critical KFERQ-like motifs (AAKFD and LIDAA) targeted for mutagenesis to generate GPX4 AAKFD and GPX4 LIDAA . (G, H) Immunoblotting (G) and quantification (H) showing that GAC induces degradation of wild-type GPX4 but not KFERQ-mutant GPX4 in HCCLM3 cells. (I) Co-IP assay demonstrating loss of interaction between HSPA8 and KFERQ-mutant GPX4 upon GAC treatment. (J, K) Overexpression of HSPA8 promotes degradation of Myc-GPX4 WT but not Myc-GPX4 AAKFD : representative immunoblots (J) and quantification (K) of GPX4, normalized to β-actin, n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Expressing, Binding Assay, Mutagenesis, Over Expression

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 1. Chemical structure of the chromone scaffold used for the virtual screening procedure (A). Chemical structure of DB987, the compound highlighted by the screening, in which the red structure represents the part of the molecule matching query (B). Chemical structure of the reference compound, PDE9 inhibitor PF-04447943 (C).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques:

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 2. Binding poses for PF-04447943 (A) and DB987 (B) in the predicted interaction pattern with PDE9. The residues at the interaction distance (<5 Å) within the binding pocket have been labeled.

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Binding Assay, Labeling

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 5. (A-B2) Immunohistochemical assessment of neuronal damage and PDE9 expression in the CA3 hippocampus of organotypic slices after treatment with KA. (A1,B1) Representative confocal images of fluorescent immunostaining of NeuN-positive neurons in area CA3 of CRL (A1) and KA (B1) slices. (A2,B2) Representative confocal images of fluorescent immunostaining of PDE9 in area CA3 of CRL (A2) and KA-treated slices (B2). (A, B) Merge of the previous images. All images were captured with a 20× objective. Scale bar: 100 μm. (C-C2) Magnifications of the framed areas of the corresponding slice shown in B, B1, and B2. (C1) Immunostaining of NeuN showing the presence of damaged neurons with an elongated, shrunk cytoplasm. (C2) Immunostaining of PDE9 showing the expression of the enzyme in many CA3 pyramidal neurons (open arrows). (C) Merge of the two previous images (open arrows indicate neurons positive for PDE9 immunostaining). Scale bar: 25 μm. (D) Quantitative analysis of PDE9 immunostaining in CA3 SP (CRL n = 10 and KA n = 9). Statistical analysis: Student’s t-test: **P < 0.01 KA vs CRL. (E-F3) Representative confocal images of triple immunofluorescent labeling of neurons (NeuN, red), PDE9 (green), and astrocytes (GFAP, blue) in CA3 of KA-treated slices captured with a 40× objective. Open arrows indicate PDE9-positive pyramidal neurons. No colocalization with astrocytes was found. Scale bars: 70 μm (E) and 15 μm (F-F3).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Labeling

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 7. Qualitative and quantitative analyses of the effects of PDE9 inhibitors in rat organotypic hippocampal slices under normal conditions or exposed to KA. (A) Hippocampal slice under normal conditions (background PI fluorescence), (B) slice exposed to 10 μM PF for 24 h, (C) slice exposed to 10 μM DB987 for 24 h, (D) slice exposed to 5 μM KA for 24 h displaying intense PI labeling in the CA3 subregion, and (E,F) CA3 damage induced by KA was attenuated by the presence of 1 μM PF and DB987. (G) PDE9 inhibitors alone did not induce side effects. (H) PDE9 inhibitors significantly attenuated CA3 damage in a dose-dependent manner. Bars represent the mean ± SEM of at least five experiments run in quadruplicate. **p < 0.01 and ***p < 0.001 vs KA (one-way ANOVA plus Dunnett’s test).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE142102 ( n = 226) dataset of TNBC patients (Pearson correlation coefficient r = 0.3245, P < 0.0001). ( B ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE103091 ( n = 238) dataset of TNBC patients (Pearson correlation coefficient r = 0.2120, P < 0.001). ( C ) Western blot showing SDCBP, BACH1, and HO-1 protein expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells. ( D ) The expression levels of SDCBP and BACH1 protein in Fig. EV1C were quantified using densitometry and normalized to the housekeeping protein α-tubulin ( n = 3). ( E ) Real-time qPCR showing SDCBP and BACH1 mRNA expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells ( n = 3). Quantitative data were normalized to β-actin expression. ( F ) Western blot showing SDCBP and HO-1 protein expression in MDA-MB-231 cells transfected with scramble or BACH1 siRNA. ( G ) Left, western blot showing the protein expression of SDCBP in the scramble and in several SDCBP-KO MDA-MB-231 subclones generated using CRISPR-Cas9 system; Right, real-time qPCR showing the SDCBP mRNA expression in scramble and in SDCBP-KO MDA-MB-231 subclones ( n = 3). ( H ) Real-time qPCR showing the mRNA expression of BACH1 in MDA-MB-231 cells, in scramble and in SDCBP-KO MDA-MB-231 subclone#2 and subclone#12 ( n = 3). ( I ) Immunofluorescence staining was used to visualize SDCBP (green color) and BACH1 (red color) in scramble and in SDCBP-KO MDA-MB-231 cells. DAPI (blue color) was used to stain the nucleus ( n = 3); Representative confocal immunofluorescence images are shown. Scale bar = 20 µm. ( J ) Western blot showing BACH1 and HO-1 protein expression in 4T1 cells infected with scramble or adenoviral SDCBP shRNA. ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1, NQO1 , and GLCL ) in 4T1 cells transfected with scramble or SDCBP siRNA ( n = 3); mRNA expression of KEAP1 was the negative control. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( D , E , G , H ) or two-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Expressing, Western Blot, Transfection, Generated, CRISPR, Immunofluorescence, Staining, Infection, shRNA, Negative Control

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunohistochemistry staining against the SDCBP and BACH1 protein in human TNBC-derived tissue microarray sections ( n = 78). Representative images showing the co-expression of SDCBP and BACH1 in the same section. Normal breast cancer tissues were considered as the negative control. Scale bar = 20 µm. ( B ) Pearson correlation coefficient (r = 0.5772, P < 0.0001) between SDCBP and BACH1 expression in ( A ). ( C ) Western blot showing BACH1 and HO-1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( D ) Real-time qPCR showing BACH1 mRNA expression in Hs578T cells transfected with a control vector or a Myc-SDCBP-expressing vector ( n = 3). ( E ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1 , and CXCR4 ) in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( F ) Western blot showing BACH1 protein expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA. ( G ) Real-time qPCR showing BACH1 mRNA expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA ( n = 3). ( H ) Left, Representative images of immunofluorescence staining to visualize SDCBP ( green color ) and BACH1 ( red color ) expression in MDA-MB-231 cells transfected with a scramble siRNA or SDCBP siRNA. DAPI ( blue color) was used to stain the nucleus ( n = 3); Scale bar = 50 µm. Right, fluorescence levels of SDCBP and BACH1 were quantified based on their spectral densities. ( I ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1, MMP13 , and CXCR4 ) in MDA-MB-231 cells transfected with scramble siRNA or SDCBP siRNA ( n = 3). ( J ) Western blot showing SDCBP and BACH1 protein expression in scramble control and two SDCBP-KO MDA-MB-231 clones (KO#2, KO#12) generated using CRISPR-Cas9 system ( n = 3). ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated metastatic genes ( HK2 , MMP1 , CXCR4, GAPDH , and VEGF ) in scramble control and SDCBP-KO MDA-MB-231 cells. ( L ) The reconstitution of SDCBP recovers BACH1 protein expression in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1 protein expression in scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector. The arrows indicate the endogenous (Endo) and exogenous (Exo) SDCBP. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( D , E , G , I ), two-way ANOVA ( H ), or one-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Microarray, Expressing, Negative Control, Western Blot, Transfection, Control, Plasmid Preparation, Infection, shRNA, Immunofluorescence, Fluorescence, Clone Assay, Generated, CRISPR, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA. ( B ) Colony formation of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . Migration of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . ( D ) Western blot showing SDCBP and Flag-BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector. Cell proliferation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( G ) Wound closure of scratched MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( H ) Schematic of various SDCBP mutant constructs generated using the Myc-SDCBP plasmid. ( I ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with the indicted SDCBP constructs. Right, quantification of BACH1 levels using densitometry ( n = 3). ( J ) Top, schematic of the PDZ1 construct. Bottom, western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP or Myc-SDCBP_PDZ1 plasmid. ( K ) Migration of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( L ) Colony formation of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( M ) Tumor volumes from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 5 or 7 mice/group). ( N ) Tumor weights in Fig. 2M ( n = 7 mice/group); See also Fig. . ( O ) Tumor volumes from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group); See also Fig. – , . Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( B , , I , K , L , N ), two-tailed Student’s t test ( , F , O ), or two-way ANOVA ( G , M ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Migration, Mutagenesis, Construct, Generated, Injection, Control, Stable Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing the expression of SDCBP and Flag-SDCBP in Fig. . ( B ) Tumor weights in Fig. ( n = 5 mice/group). ( C ) Tumor volume in Fig. ( n = 5 mice/group). ( D ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 7 mice/group). Representative images of IHC staining are shown. Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( E ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, or SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group). Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( F ) TCGA data analysis showing association between SDCBP mRNA expression and overall survival ( n = 392) and lymph node status ( n = 98) of TNBC patients. ( G ) TCGA data analysis showing association between BACH1 mRNA expression and overall survival ( n = 2032) and lymph node status ( n = 98) of TNBC patients. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( B ) or two-tailed Student’s t test ( C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Isolation, Injection, Control, Stable Transfection, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without MG132 proteasome inhibitor. ( B ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( C ) Left, western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( D ) Western blot showing BACH1, FBXO22, and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with HA-FBXO22-expressiong vector with or without Myc-SDCBP-expressing vector. ( F ) In vitro ubiquitylation assay of the recombinant human BACH1 protein mediated by the FBXO22 complex. Active recombinant human UbcH5a protein was used as the E2 ubiquitin-conjugating enzyme for FBXO22 complex-mediated BACH1 degradative polyubiquitylation. ( G ) In vivo ubiquitylation assay showing the decrease in the K48-linked polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. ( H ) In vivo ubiquitylation assay showing the increase in the K48-linked polyubiquitylation of BACH1 by SDCBP KD in MDA-MB-231 cells transfected with the indicated plasmids. ( I ) In vitro ubiquitylation assay showing the inhibitory effect of SDCBP on the polyubiquitylation of BACH1 mediated by the FBXO22 complex. Active recombinant human protein UbcH5a and immunocomplex FBXO22 were added as described above. Recombinant human BACH1 and recombinant human SDCBP proteins were added at ratios of 1:1 (+) and 1:2 (++). Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( B , C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, In Vitro, Ubiquitin Assay, Recombinant, Ubiquitin Proteomics, In Vivo, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Real-time qPCR showing the mRNA expression of BACH1-regulated ETC genes ( NDUFA4 , NDUFA4L2 , NDUFC2 , and COX6B2 ) in scramble and SDCBP-KO MDA-MB-231 cells ( n = 3); See also Appendix Fig. S , . Real-time qPCR showing the mRNA expression of NDUFA4 and COX6B2 in scramble, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells transfected with SDCBP ( n = 3). ( C ) Western blots showing the expression of mitochondrial proteins NDUFA4 and COX6B2 in MDA-MB-231 cells transfected with SDCBP siRNA in the presence or absence of FLAG-BACH1-expressing vector. ( D ) ChIP-qPCR showing BACH1 enrichments in the promoter regions of NDUFA4 and COX6B2 in the scramble and SDCBP-KO MDA-MB-231 cells. Quantitative data were normalized to IgG binding expression ( n = 3); See also Appendix Fig. S . ( E ) Left, flow cytometry histogram showing the mitochondrial membrane potentials using TMRE (tetramethylrhodamine ethyl ester) staining in MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after the indicated treatments. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone). Right, quantification of TMRE fluorescence intensity ( n = 3). ( F ) Left, immunofluorescence staining and confocal imaging of the fluorescent signals for TMRE (orange-red color) in the scramble control and SDCBP-KO MDA-MB-231 cells after incubation with TMRE. DAPI (blue color) was used to stain the nucleus ( n = 7); Representative confocal images are shown; scale bars = 20 µm and 5 µm. Right, fluorescent levels of the TMRE were quantified based on their spectral densities. ( G ) Representative images of immunohistochemistry staining against the NDUFA4, BACH1, and SDCBP proteins showing a negative correlation between the expression of NDUFA4 and SDCBP in the same sections of TNBC tumor tissues. Scale bar = 20 µm. ( H ) Pearson correlation coefficients between SDCBP and NDUFA4 protein expression ( n = 64), and between BACH1 and NDUFA4 protein expression ( n = 60) in Fig. 5G. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A , D , F ), two-tailed Student’s t test , or one-way ANOVA ( E ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Flow Cytometry, Membrane, Staining, Fluorescence, Immunofluorescence, Imaging, Control, Incubation, Immunohistochemistry, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Cell proliferation of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with metformin for the indicated periods of time ( n = 3). ( B ) Cell viability of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin for 96 h ( n = 5). ( C ) Colony formation for MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin. Colony numbers were counted and converted to percentages by normalizing with the control groups ( n = 3). ( D – F ) Effect of the indicated treatment on 4T1 tumor growth. Tumor growth was monitored in BALB/c mice bearing 4T1 cells after mammary fat-pad injections. When the average tumor volumes reached 100 mm 3 , the mice ( n = 7 mice/group) were administered with 100 mg/kg metformin (once a day) and/or adenoviral SDCBP shRNA (1 × 10 9 PFU/mice). Black arrows indicate the day of adenoviral SDCBP shRNA injection. Final tumor volume ( E ) and weight ( F ) are shown. ( G ) Immunohistochemistry staining against SDCBP, BACH1, Ki67, NDUFA4, and COX6B2 protein in 4T1 tumors from BALB/c mice in Fig. 6A. Representative images of the IHC staining are shown. Scale bar = 50 µm for low (left) and high (right) magnification. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A – C ), one-way ANOVA ( E ), or two-tailed Student’s t test ( F ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Transfection, Control, shRNA, Injection, Immunohistochemistry, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: Schematic showing the novel oncogenic roles of SDCBP in promoting aggressiveness and mitochondrial inhibitor resistance in TNBCs. An abundance of SDCBP stabilizes the BACH1 protein by blocking E3 ubiquitin ligase SCF FBXO22 complex-targeted BACH1 for degradative ubiquitination. Mechanistically, SDCBP binds to different members of the SCF FBXO22 complex, including SKP1 and FBXO22, via its PDZ1 domain and induces SCF FBXO22 complex disassociation, suggesting that SDCBP is a key adapter regulating the activity of the E3 ubiquitin ligase SCF FBXO22 complex in the proteasomal pathway. SDCBP-induced BACH1 accumulation upregulates several pro-metastatic genes and downregulates numerous mitochondrial ETC genes, resulting in tumor progression and high resistance to metformin treatment in TNBCs. Targeting SDCBP switches the SCF FBXO22 complex to degrade BACH1 protein via the proteasome, reducing tumor aggressiveness and boosting the anti-tumor effect of metformin administration in TNBCs. .

Article Snippet: The IP lysates were added with UbcH5A recombinant protein (CAT#ab269096, Abcam, USA), BACH1 recombinant protein (CAT#TP321628, OriGene, USA), and SDCBP recombinant protein (CAT#15640-H07E, Sino Biological, USA) following the indicated experimental designs, then performed invitro ubiquitination reactions by using Ubiquitylation Assay Kit (CAT#ab139647, Abcam, USA), followed by immunoblotting analysis according to manufacturer’s instructions.

Techniques: Blocking Assay, Ubiquitin Proteomics, Activity Assay

Journal: PLoS ONE

Article Title: Crosstalks of the PTPIP51 interactome revealed in Her2 amplified breast cancer cells by the novel small molecule LDC3/Dynarrestin

doi: 10.1371/journal.pone.0216642

Figure Lengend Snippet: Antibody list.

Article Snippet: IκBα , Recombinant Human IκB alpha/NFKBIA protein 02 , Mouse monoclonal , MM02 , 1:100 , Sino Biological Inc., North Wales, PA, USA Cat.# 12045-H07E.

Techniques: Recombinant, Purification

Journal: bioRxiv

Article Title: A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease

doi: 10.1101/555557

Figure Lengend Snippet: (a) scHPF was used to factorize scRNA-seq profiles of each tissue and blood sample independently after merging resting and activated T cells. Each matrix element in the heatmap is the pairwise Pearson’s correlation coefficient between the gene scores for a pair of factors computed across a set of high-and low-scoring genes (see Online Methods). The resulting modules were named based on the identities of their highest scoring genes (Supplementary Table 7) and the resting vs. activated status of the highest scoring cells. Lower color bars show the tissue and donor of origin, and the CD4/CD8 bias of cell scores for each factor. (b) Diffusion maps generated from scHPF factors (see Online Methods) for CD4 + and CD8 + T cells from each tissue and blood sample after merging resting and activated T cells with each cell colored by expression of IL2RA . (c) Same as (b) but colored by expression of NME1 . (d) Same as (b) but colored by expression of IFIT3 . (e) Same as (b) but colored by expression of IFNG .

Article Snippet: At indicated time points, we performed intercellular staining of NME1 (11615-H07E; Sino Biological) using a Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) for fixation and permeabilization of cells according to manufacturer’s instructions.

Techniques: Diffusion-based Assay, Generated, Expressing

Journal: bioRxiv

Article Title: A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease

doi: 10.1101/555557

Figure Lengend Snippet: (a) Expression of NME1 and IL2RA mRNA by blood CD4 + or CD8 + T cells after stimulation with anti-CD3/anti-CD28 antibodies by qPCR. Data shown as mean fold-change (±SEM) relative to unstimulated CD4 + or CD8 + T cell controls (dotted line) from 4 individuals (independent experiments). Statistical analysis between stimulated and unstimulated cells (black *) or CD4 + and CD8 + T cells (red *) made by two-way ANOVA with Sidak test for multiple comparisons. (b) Intracellular NME1 protein expression by blood T cells after stimulation for indicated timepoints (red) compared to unstimulated (black) and isotype control (gray). Bottom row: CD25 and NME1 expression by proliferating CD3 + T cells after 5 days of stimulation. Data are representative of 4 individuals. (c) Expression of IFIT3 mRNA in blood T cells by qPCR after TCR-stimulation, shown as mean fold-change (±SEM) relative to unstimulated controls (dotted line) for four individuals. Two-way ANOVA with Sidak test for multiple comparisons was used for statistical comparisons (black *, stimulated versus unstimulated) or (red *, CD4 + versus CD8 + T cells). (d) IFIT3 or NME1 mRNA expression in CD4 + T cells after culture with anti-CD3/anti-CD28 or IFNα2 (1000 units/mL) +/-type I IFN neutralizing antibody cocktail or (e) IFNγ (10 ng/mL) +/-anti-IFNγ/anti-IFNγR1 antibodies (1 ug/mL each), shown as mean fold-change (±SEM) relative to unstimulated controls (dotted line) for 3 individuals. Statistical comparisons made by two-way ANOVA. For all panels: “ns” denotes not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: At indicated time points, we performed intercellular staining of NME1 (11615-H07E; Sino Biological) using a Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) for fixation and permeabilization of cells according to manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease

doi: 10.1101/555557

Figure Lengend Snippet: (a) Merged UMAP embedding for the entire healthy T cell scRNAseq dataset including resting and activated tissue T cells (two donors) and blood T cells (two individual) colored by sample source, donor, resting/activated condition, CD4/CD8 status (CD4-enriched, green; CD8-enriched, purple), and CCL5 expression indicating TEM cells. (b) Merged UMAP embedding for the entire dataset overlaid with contour plots indicating kernel density estimates for the projection of T cells derived from organ/blood donors (column 1), non-small cell lung cancer (NSCLC) tissue (column 2), colorectal cancer (CRC) tissue (column 3), breast cancer (BC) tissue (column 4), and melanoma (MEL) tissue (column 5). Note that these probability densities can be compared within each projection, but cannot be quantitatively compared across projections. (c) Same as b) but overlaid with a two-dimensional hexbin histogram for each projection. Histograms have been normalized to account for differences in cell numbers across datasets and therefore can be compared quantitatively across projections. (d) Individual cells in the UMAP embedding (column 1) for the entire healthy T cell dataset and UMAP projections (columns 2-5) for NSCLC, CRC, BC, and MEL tissue T cells colored by expression of CD4 , CD8A , FOXP3 (Treg marker), CXCR6 (TRM marker), IFIT3 (IFN response marker), NME1 (activation marker), PRF1 (cytotoxic marker), and IFNG . Expression values are normalized for quantitative comparison within each dataset (i.e., column), but not across datasets.

Article Snippet: At indicated time points, we performed intercellular staining of NME1 (11615-H07E; Sino Biological) using a Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) for fixation and permeabilization of cells according to manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Marker, Activation Assay