Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Table 1 Sequences of primers used in this study

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 is upregulated in the process by which Wnt4 promotes neuronal differentiation in NSCs (A,B) RNA sequencing of NSCs treated with Wnt4 for 3 days. (A) Volcano plots of differentially expressed genes (DEGs) in NSCs treated with Wnt4 in comparison with the untreated group. (B) Heatmap of DEGs of NSCs treated with Wnt4 in comparison with the untreated group. (C,D) RT-qPCR and western blot analysis of Fut9 expression in NSCs stimulated with Wnt4 for 3 days. (E) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: RNA Sequencing, Comparison, Quantitative RT-PCR, Western Blot, Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Wnt4 upregulates Fut9 expression through the β-catenin signaling pathway (A) GO pathway enrichment analysis of the differentially expressed genes between the NT group and Wnt4 group. (B,C) RT-qPCR and western blot analysis of Fut9 expression in NSCs treated with a specific pathway inhibitor and then stimulated with Wnt4 for 3 days. (D) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group; #P<0.05 compared with the Wnt4 group. IWR-1: Wnt/β-catenin inhibitor.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Overexpression of Fut9 promotes neuronal differentiation (A,B) mRNA and protein expression levels of Fut9 in the NT, LV-Con, and LV-Fut9 groups. (C) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con, and LV-Fut9 groups. Scale bar: 100 μm. (D,E) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (F,G) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con, and LV-Fut9 groups. (H) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the nontreatment group and LV-Con group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Marker, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 rescues the negative effect of Notch signaling to promote neuronal differentiation (A) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. Scale bar: 100 μm. (B, C) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (D,E) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. (F) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+Jag1 group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Immunofluorescence, Staining, Marker, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 inhibits the furin enzyme activity of S1 cleavage to suppress the activation of the Notch signaling pathway (A) Western blot analysis of Notch1 and NICD expression in NSCs treated with furin. (B) Quantification of Notch1 and NICD expression by western blot analysis. (C) Furin-like enzyme activity was measured in NSCs in different groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group. (D,E) RT-qPCR and western blot analysis of Hes1 and Hes5 in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. (F) RT-qPCR analysis of neural transcription factor expression in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+furin group.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Activity Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

doi: 10.3724/abbs.2023138

Figure Lengend Snippet: Fut9 promotes functional recovery and tissue repair (A) Images showing hind limb climb from Sham, SCI, LV-Con and LV-Fut9 groups at the eighth week postinjury; white arrows point to the hind limb. (B) BBB scores of the different groups. (C) H&E, MRI and Nissl staining analyses of the spinal cord in different groups. Sections at 2 mm rostral and caudal to the lesion epicenter were counted for each rat. (D,F) Quantification of H&E, MRI and Nissl staining analyses in different groups. Data are presented as the mean±SD. *P<0.05 compared with the sham group; #P<0.05 compared with the SCI group. (G) Immunofluorescence staining of the spinal cord in different groups. Scale bar: 50 μm. (H) Quantification of immunofluorescence staining. Data are presented as the mean±SD from two independent experiments. **P<0.001.

Article Snippet: The lentiviral vectors carrying green fluorescent protein (GFP) with a sequence specifically overexpressing the Fut9 gene were constructed by GeneChem (Shanghai, China).

Techniques: Functional Assay, Staining, Immunofluorescence

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet: IFN-stimulated, Ly6E (hi) neutrophils mark response to αPD1 in 4T1 breast cancer 10X scRNA-seq was performed on GR1 + cells obtained from parental (4T1 P ) (non-responsive) and mutagenized (4T1 M ) (responsive) 4T1 breast cancer tumors (n = 3 mice pooled/group). (A) UMAP plot of 2886 filtered, GR1 + neutrophils (4T1 P = 681 cells, 4T1 M = 2185 cells), with cells colored based on differential abundance score. Two significantly enriched, cellular neighborhoods (dotted lines) are highlighted (see also Figure S2 C). The top 10, most significant marker genes of each neighborhood are listed (FDR < 0.001, log 2 fold-change > 1.5). Monocytic cells (not shown) were discarded from the analysis (see: Figure S2 ). (B) Trajectory analysis for 12 distinct, GR1 + granulocytic clusters. Solid black line = trajectory lineages, which form the basis of the pseudotemporal ordering as inferred by partition-graph based abstraction (PAGA). Black arrows = simplified RNA-velocity (for raw data, see Figure S2 D). (C) Top: A histogram of binned cell frequencies as a function of aligned pseudotime. Smoothed distributions, generated by loess regression, are overlaid. Significance was assessed by means of two-sample KS-test. Bottom: A heatmap of normalized, binned enrichment scores for all gene modules that display a significant association with pseudotime (FDR < 0.01). Only gene-modules common to both lineages are shown. (D) Boxplots showing the concentration of IFNγ, TNFα and IFNα within untreated 4T1 tumor lysates (n = 4-5 mice/group). (E) Binned, normalized expression of Ly6E. Data were imputed for visual clarity. (F and G) Frequencies of Ly6E (hi) neutrophils, as determined by flow cytometry (n = 5-10 mice/group), in 4T1 tumors (F); and the blood of 4T1 bearing mice (G); For the gating strategy see Figure S3 A. In (D, F, and G), significance was assessed by means of a one-way Mann-Whitney test (NS, p > 0.01; ∗ , p < 0.01; ∗∗ , p < 0.001, ∗∗∗ , p < 0.0001).

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Marker, Generated, Concentration Assay, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet: Ly6E (hi) neutrophils sensitize non-responding 4T1 tumors to αPD1 treatment (A) Schematic of adoptive transfer. Isolated GR1 + cells are treated in vitro with IFNγ/α, inducing a Ly6E ( hi ) -like state, characterized by secretion of effector molecules, and injected into BALB/c mice bearing parental, non-responsive 4T1 breast tumors. (B) Frequency of Ly6E (hi) neutrophils following exposure of GR1 + cells to IFNγ, IFNα or both, as determined by flow cytometry (n = 3 mice/group). Significance was assessed by means of a one-way ANOVA and Tukey’s post-hoc HSD test (NS, p > 0.01; ∗∗ , p < 0.001; ∗∗∗ , p < 0.0001). (C) A heatmap comparing normalized, log 2 -fold changes from RT-qPCR (treated [+IFNγ/α] vs. untreated control GR1 + cells) and scRNA-seq (Ly6E (hi) neutrophils vs. all remaining neutrophils) (n = 7 biological repeats/group). SC = scRNA-seq. μm = averaged RT-qPCR values. (D) Averaged tumor growth profiles for mice bearing parental, non-responsive 4T1 breast tumors treated with either a monotherapy (control IgG or αPD1) or a combined therapy, with GR1 + or Ly6E (hi) neutrophils, as specified (n = 6 mice/group). A time-course of the adoptive transfer is depicted in ( Figure S4 A). Raw data are available in ( Figure S4 B). Treatment was initiated at a tumor size of ∼50 mm 3 (arrow). Significance was assessed by means of two-sample KS-test ( ∗∗∗ , p < 0.0001).

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Adoptive Transfer Assay, Isolation, In Vitro, Injection, Flow Cytometry, Quantitative RT-PCR

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet: Tumor-intrinsic STING activity induces the Ly6E (hi) phenotype and in-turn supports activation of effector T cells (A) Density plots of dsDNA levels in cultured 4T1 P and 4T1 M cell-lines, as determined by α-dsDNA staining and flow cytometry. dsDNA levels were quantified relative to an unstained, IgG2a isotype control (CTRL) (n = 5 biological repeats/group). (B) Densitometry quantification of western blots (see Figure S5 A) for STING-pathway related proteins in 4T1 P and 4T1 M tumor lysates (n = 3–4 biological repeats/group). Each protein was normalized relative to an actin control. (C) Isolated GR1 + cells were cultured in vitro with conditioned media generated from 4T1 P (P) or 4T1 M (M) tumors in the presence or absence of the STING-inhibitor H151 or αIFNR-α/γ, and the frequencies of Ly6E (hi) neutrophils were determined by flow cytometry (n = 6 biological repeats/group). CTRL = GR1 + cells only. (D and E) Conditioned media was generated from GR1 + cells or IFNαγ-induced Ly6E (hi) neutrophils, and subsequently assayed on a cytokine array (n= 3 mice pooled/group). Hyper-geometric, over-representation tests and the Gene Ontology (GO) database were used to determine enriched pathways for Ly6E (hi) neutrophils (D); and GR1 + cells (E). Only differentially expressed proteins with a log 2 FC > 0.35 were included and only significant pathways (FDR < 0.01) are shown. (F) Isolated CD8 + T cells were cultured in vitro with α-IL-12b or α-IL23a neutralizing antibodies, with or without conditioned media from IFNα/γ-induced Ly6E (hi) neutrophils (L), and the levels of activated CD25 + CD8 + T cells were determined by flow cytometry (n = 5 mice/group). CTRL = CD8 + T cells only. In (B, C, and F), significance was assessed by means of a one-way ANOVA and Tukey’s post-hoc HSD test (NS, p > 0.01; ∗ , p < 0.01; ∗∗ , p < 0.001; ∗∗∗ , p < 0.0001). (G) Schematic of the proposed mechanism. Tumor-intrinsic STING activity, as induced by cytosolic dsDNA as a result of hypoxia, genomic instability and/or cell stress, transcriptionally activates an IFN response. Tumor-secreted IFNα, for example, subsequently binds to Ifnar-expressing Neutrophils in the TME, inducing the Ly6E (hi) phenotype and in-turn activation and proliferation of CD8 + T cells through IL-12b. Collectively, this supports immunotherapy response and anti-tumor activity. It is important to note that this mechanism is STING-specific, but that Type II IFNs (e.g., IFNγ)—derived from other sources or mechanisms—are also able to elicit equivalent effects, as shown in our work.

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Activity Assay, Activation Assay, Cell Culture, Staining, Flow Cytometry, Western Blot, Isolation, In Vitro, Generated, Expressing, Derivative Assay

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet: Ly6E (hi) neutrophils serve as a predictive biomarker for immunotherapy response in humans (A) UMAP plot of 11702 filtered, CD45 + cells taken from publicly available non-small cell lung cancer (NSCLC) scRNA-seq data (patient blood samples at baseline, n = 8) , with cells colored by cell type. (B) Binned UMAP plot of isolated neutrophils (dotted box in (A)), with cells colored by the extent of their enrichment for a Ly6E (hi) functional signature. The top 10, most significant marker genes of the enriched cluster (dotted lines) are listed (FDR < 0.001, log 2 fold-change > 1.5). (C) Binned, normalized expression of Ly6E. Data were imputed for visual clarity. (D and E) Frequency of Ly6E (hi) neutrophils in the blood of an independent cohort of patients with NSCLC (n = 50) (D) and skin cutaneous melanoma (SKCM) (n = 59) (E), as determined by flow cytometry. For the gating strategy see Figure S3 B. Data are stratified by RECIST categories at 3 and/or 6 months (NR = progressive disease (PD) and R = stable disease (SD), partial or complete response (P/CR)). Sample sizes are denoted for each individual group. Significance was assessed by means of a one-way Mann-Whitney test ( ∗∗∗ , p < 0.0001). (F) Smoothed area under the curve (AUC)-receiver operating characteristics (ROC) plots for Ly6E (hi) neutrophils (95% CIs: 0.855–0.9705 (NSCLC - LC), 0.7913–0.9606 (Melanoma - MN)), absolute neutrophil count (Abs Neut) (95% CIs: 0.534–0.9328 (in NSCLC)) and tumor PDL1 IHC (95% CIs: 0.3554–0.9338 (in NSCLC)) in our cohort of patients (NR vs. R). Confidence intervals were determined using 1,000 stratified, bootstrap replicates.

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Biomarker Assay, Isolation, Functional Assay, Marker, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet: A Ly6E (hi) neutrophil-derived gene signature outperforms pre-existing biomarkers in the prediction of immunotherapy response (A) Bulk RNA-seq expression profiles were obtained from 1,440 publicly available samples from 11 datasets across 6 cancer types , , , (see ) and scored for a 15-gene Ly6E (hi) -signature (Neut IFN -15) (top) or a previously published 6-gene IFNγ-signature (bottom). A heatmap of median, normalized enrichment scores for each dataset is shown and significant differences between groups were tested (NR vs. R). Samples were taken either pre-treatment (PRE) or post-treatment (POST). Raw data are available in Figure S10 . BLCA = urothelial bladder cancer; GBM = glioblastoma multiforme; NSCLC = non-small cell lung cancer; RCC = renal cell carcinoma; SKCM = skin cutaneous melanoma; STAD = stomach adenocarcinoma. Significance was assessed by means of a one-way Mann-Whitney test (NS, p > 0.01; ∗ , p < 0.01; ∗∗ , p < 0.001, ∗∗∗ , p < 0.0001). (B) Smoothed area under the curve (AUC)-receiver operating characteristics (ROC) plots for total tumor mutation burden (tTMB) (95% CIs: 0.4865-0.6722), Age (95% CIs: 0.4374-0.5766), PDL1 immunohistochemistry (IHC) (95% CIs: 0.5534-0.7172), STK11 mutational status (95% CIs: 0.5246-0.6874), KEAP1 mutational status (95% CIs: 0.5334-0.7085), IFNγ-6 signature scores (95% CIs: 0.6253-0.7561) and Ly6E (hi) Neut IFN -15 signature scores (95% CIs: 0.7714-0.9105) in data from the OAK NSCLC study (NR vs. R). Confidence intervals were determined using 1,000 stratified, bootstrap replicates.

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Derivative Assay, RNA Sequencing Assay, Expressing, MANN-WHITNEY, Mutagenesis, Immunohistochemistry

Journal: Cancer Cell

Article Title: Interferon-stimulated neutrophils as a predictor of immunotherapy response

doi: 10.1016/j.ccell.2023.12.005

Figure Lengend Snippet:

Article Snippet: Next, lentiviral particles were generated by co-transfecting HEK-293FT cells with packaging (psPAX2, Addgene Plasmid #12259) and envelope (pMD2.G, Addgene Plasmid #12259) plasmids together with pXPR_053 (control vector, Addgene Plasmid # 113591) or pXPR_053 vector containing gRNA specific for the Ly6E gene in StemSpam SFEM II media (Stemcell technologies, Cat# 09605).

Techniques: Recombinant, In Vivo, Modification, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Selection, Labeling, Fluorescence, Purification, SYBR Green Assay, Cell Isolation, RNA Sequencing Assay, Plasmid Preparation, Software

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of characteristic genes in vivo (A) Relative expression levels of DDR-related genes in control and sepsis groups (n=4 in each group). (B) Relative expression levels of ARL4C in control, sepsis, sepsis+Ad-shNC, and sepsis+Ad-shARL4C group (n=8 in each group). (C) Expression levels of inflammatory factors (IL-1β, TNF-α, IL-10, and IL-18) in the peripheral blood of rat with sepsis+Ad-shNC and sepsis+Ad-shARL4C (n=8 in each group). (D) Survival status of rats in each group (n=10 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS, no significant difference.

Article Snippet: For knockdown experiments, shRNA plasmids targeting ARL4C and negative control shRNA were procured from RiboBio (Guangzhou, China), and the specified shRNA lentiviral vectors were generated in 293 T cells.

Techniques: Biomarker Discovery, In Vivo, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of ARL4C in vitro . (A) Flow cytometry detected the apoptosis rate in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group) (B) Flow cytometry detected the ROS production in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group), *** p < 0.001, **** p < 0.001. NS, no significant difference.

Article Snippet: For knockdown experiments, shRNA plasmids targeting ARL4C and negative control shRNA were procured from RiboBio (Guangzhou, China), and the specified shRNA lentiviral vectors were generated in 293 T cells.

Techniques: Biomarker Discovery, In Vitro, Flow Cytometry, Control