Journal: bioRxiv

Article Title: β2-Adrenergic Biased Agonist Nebivolol Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner

doi: 10.1101/2024.09.08.611829

Figure Lengend Snippet: Effect of A) β1AR antagonist (metoprolol) and B) non-selective βARs antagonist (bupranolol) on the production of IL-17A on memory Th cells. Activated memory Th cells were blocked for 30 minutes with metoprolol or bupranolol before treatment with nebivolol and incubated for five days. IL-17A was measured in cell culture supernatants. Pooled data are expressed as the Mean ± SEM of four independent biological experiments. Effect of CRISPR/Cas9-mediated knockout of β2AR on nebivolol treatment in activated memory Th cells. Stimulated memory Th cells were electroporated with a CRISPR/Cas9 system targeted by multi-sgRNA against β2AR or non-targeting genes. C) The expression of ADRB2 at mRNA levels as verification in non-electroporated, non-targeting and β2 multi-sgRNA conditions has shown as the relative amounts normalized to housekeeping RNA and compared to a control set to 1.0 (dotted line). The data of this figure is representative of three independent biological experiments. Three days after electroporation, memory Th cells were cultured with or without nebivolol for another 5 days and D) A representative of IL-17A levels in cell culture supernatants in both control and knock-out β2AR conditions was shown. ANOVA was followed by Tukey’s multiple comparisons tests.

Article Snippet: According to published protocols ( , ), before electroporation briefly, 100 pmol of optimized multi single-guide RNA (sgRNA) (payload sequences attached in supplementary table1) (Synthego, USA) and 50 pmol of Cas9 (Thermofisher, CA) per 1 million cells were mixed in 5 μL of Resuspension Buffer R and incubated at room temperature for 10 minutes to form the Cas9-ribonucleoprotein (RNP) complex.

Techniques: Incubation, Cell Culture, CRISPR, Knock-Out, Expressing, Control, Electroporation

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Relative expression of IEC markers from different branches, including enteroendocrine cells ( Neurog3 , Insm , and Pax4 ), secretory lineage ( Muc2 , Gfi1 , Foxa2 , Reg3a , and Lyz ), M cells ( Spib ), Tuft cells ( Dclk ), stem cells ( Olfm4 , Lgr5 ), and absorptive cells ( Si ) in mouse organoids with rSPINK4 stimulation. The degree of color depth indicates the abundance of expression positively. b Levels of phosphorylated and total EGFR in NCM460 and HT-29 cells with 5 ng/mL rSPINK4 stimulation for 0–60 min. c Direct downstream molecules of the EGFR pathway detected using western blotting with rSPINK4 stimulation for 0–60 min. d Typical fluorescent images of intestinal organoids from WT and KO mice including MUC2 (green), E-cadherin (red), and DAPI (blue), with rSPINK4 (100 ng/mL) and AG1478 (10 μM) stimulation under inflammatory conditions (50 ng/mL TNF-α treatment). e Quantitative analysis of weight loss with SPINK4 and gefitinib intervention following DSS administration in the control group drinking water (control, n = 4); DSS and PBS administration group (DSS + PBS, n = 6); DSS and rSPINK4 administration group (DSS + rSPINK4, n = 5); and DSS, rSPINK4, and gefitinib administration group (DSS + rSPINK4 + gefitinib, n = 4). f Representative canonical endoscopic images with the lesion circled with white arrows. g Quantification of colonic length. h The levels of the phosphorylated and total EGFR were determined in WT and KO mouse tissues (upper panel) and rSPINK4 treatment group (lower panel). Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( h ). Besides, Kruskal–Wallis test ( h ) and one-way ANOVA ( b , e , g ) were performed for multiple comparison; n = 3–5 biologically independent experiments ( b , g , h ). Source data are provided as a Source Data file.

Article Snippet: Transient transfection was achieved via single-guide RNA (sgRNA) of SPINK4 and EGFR synthesized by OBiO Technology.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a The FLAG-SPINK4 protein, which exists in the concentrated medium of SPINK4- overexpressing cells, is involved in the EGFR protein from cell lysis combined with EGFR antibody. IgG is the contrast to EGFR antibody. b The binding test of rSPINK4 protein and EGFR in membrane extraction under the incubation in vitro. c rSPINK4 coprecipitates with rEGFR (extracellular part) mutually in the in vitro pull-down assay controlled by IgG. d Representative immunofluorescent image of rSPINK4 and EGFR localization (EGFR: green, rSPINK4: red, DAPI: blue). e The curve of released heat and change in △H are performed when the rEGFR-His protein (6 μM) is titrated by rSPINK4-His protein (30 μM) via the ITC assay. f The pull-down assay of rEGF and rEGFR (extracellular part) in a SPINK4-dose-dependent manner. The concentration of rSPINK4 is shown. g A typical binding curve between biotinylated EGF and EGFR was performed using AlphaLISA binding assay. h Image profile of the interaction between rSPINK4 and the deleted extracellular domain of EGFR (△1: the deletion of 57–168 aa, △2: the deletion of 177–338 aa, △3: the deletion of 361–481 aa, △4: the deletion of 505–637 aa, ED: whole extracellular EGFR, NC: vector). i Interaction between mutant rSPINK4 (wt: whole sequence of SPINK4, mut1: mutation sites: C65A, C68A, and C86A, mut2: mutation sites: Q48A and M49A, NC: vector) and extracellular EGFR in vitro. j Predictive interactive model of SPINK4 and extracellular EGFR (pink: SPINK4, the tail is at N-terminal; red: EGFR subdomain including 57–168 aa; green: EGFR subdomain including 177–338 aa; yellow: EGFR subdomain including 361–481 aa; orange: EGFR subdomain including 505–637 aa). Data are presented as the mean ± SEM. The Statistical test was two-sided using one-way ANOVA ( g ); n = 6 biologically independent experiments ( g ). Source data are provided as a Source Data file.

Article Snippet: Transient transfection was achieved via single-guide RNA (sgRNA) of SPINK4 and EGFR synthesized by OBiO Technology.

Techniques: Lysis, Binding Assay, Membrane, Extraction, Incubation, In Vitro, Pull Down Assay, Isothermal Titration Calorimetry, Concentration Assay, Plasmid Preparation, Mutagenesis, Sequencing

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a , b SPINK4 levels in LS174T cell supernatant stimulated with Pam2CSK4 ( a ) and carbachol ( b ). c , d Relative expression of SPINK4 with 100 μg/mL Pam2CSK4 stimulation in human intestinal organoids ( c ) and with stimulation with a lower concentration (0.01 μg/mL) in mouse intestinal organoids ( d ). e SPINK4 expression presented with MUC2, Lyz, and CgA values in the intestinal organoids from WT and cKO mice with Pam2CSK4 treatment. f Gel-forming mucin and transmembrane mucin expression when SPINK4 was overexpressed or knocked out were assessed using qRT-PCR test. OE, overexpression; NC, negative control; KO, knockout. g MUC2 expression was influenced by the intrinsic SPINK4 levels in the LS174T cells and the phosphorylated EGFR levels. OE, overexpression; NC, negative control; KO, knockout. Blank indicates no vector involved. h , i The correlation between MUC2 level with SPINK4 expression in IBD. The mRNA level was normalized to β-actin level ( h ). The Pearson correlation coefficient ( r ) was used to estimate the correlation between MUC2 and SPINK4 expression ( i ). j The apparent difference in colonic permeability following Spink4 knockout (UEA1: green, EUB338: red, DAPI: blue; white arrows: the bacteria which penetrate the inner mucus layer; red triangles: the outside of the inner mucus layer. Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( d , g ) and one-way analysis of variance (ANOVA) ( a , b , g ). Mann–Whitney U test and Kruskal–Wallis test were performed on non-normal data ( c , h ); n = 3 biologically independent experiments ( a – d , g ). Source data are provided as a Source Data file.

Article Snippet: Transient transfection was achieved via single-guide RNA (sgRNA) of SPINK4 and EGFR synthesized by OBiO Technology.

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Over Expression, Negative Control, Knock-Out, Plasmid Preparation, Permeability, Bacteria, MANN-WHITNEY

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Circulating SPINK4 level from healthy controls ( n = 64), UC ( n = 51), and CD patients ( n = 108) detected using ELISA. b SPINK4 levels between remission and active stages, based on clinical characteristics (left) or endoscopic performance (right) in the sera of IBD patients. c , d The efficiency of SPINK4 (red line), ESR (green line), CRP (blue line), and PLT (orange line) values for endoscopic ( c ) or clinical ( d ) activity assessment are shown via ROC curve. e Percentage of different level of SPINK4 in multiple intestinal locations including L1 (ileal), L2 (colonic), and L3 (ileocolonic), according to the Montreal classification. f Graphic abstract showing the involvement of extracellular SPINK4 in the regeneration of GCs via directly targeting EGFR pathway. Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( b , c ) and one-way analysis of variance (ANOVA) ( a ). Source data are provided as a Source Data file.

Article Snippet: Transient transfection was achieved via single-guide RNA (sgRNA) of SPINK4 and EGFR synthesized by OBiO Technology.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: A putative target gene SLC12A5 is associated with disrupted topologically associating domain. (A) Pearson correlation among the expression of 64 potential genes derived from Hi‐C data in TCGA glioma. (B) Biological process enrichment of 64 potential genes in GO. (C) Left: Accessibility of gene‐related enhancer peak between TCGA‐GBM and TCGA‐LGG. Right: Mean expression of each gene between TCGA glioma tissue and GTEX normal brain tissue. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, ns: no statistics. (D) Venn diagram displaying intersections of genes with differential accessibility of enhancer peak and expression level between TCGA glioma tissue and GTEX normal brain tissue. (E) Top: Specific enhancer peak signals of SLC12A5 between TCGA‐GBM and TCGA‐LGG. Bottom: Expression level of SLC12A5 between TCGA glioma tissue and GTEX normal brain tissue. (F) Altered TAD structures where SLC12A5 is located between A172 and HA1800 cell line based on Hi‐C data.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Expressing, Derivative Assay, Hi-C

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: Clinicopathological characteristics of SLC12A5 in glioma. (A) The relationship between expression of SLC12A5 and other clinical features (Grade, Histology, IDH mutation and 1p/19q status) in TCGA, CGGA1, CGGA2 and GSE16011 data sets. Statistic test: chi‐square test. (B) Expression level of SLC12A5 among CL, PN and MES subtypes in TCGA glioma. (C) Expression level of SLC12A5 between tumour and normal tissue in pan‐cancer data sets. * p < 0.05, ** p < 0.01 and *** p < 0.001, **** p < 0.0001, ns: no statistics.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Expressing, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: Prognostic value of SLC12A5 in glioma. (A) Correlation between SLC12A5 and overall survival of glioma patients in six cohorts. (B) Meta‐analysis of multivariate cox results from three data sets using random effect and fixed effect model.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: Characterization of genomic alterations, transcriptome features and drug response correlated with SLC12A5. (A) Landscape of somatic mutation between SLC12A5 high‐expression group and low‐expression group. (B) Network of enriched GO categories based on DEGs between these two groups ( Q value <0.05). Normal Enrichment score (NES) >0 indicates upregulated processes in SLC12A5 high‐expression group. (C) KEGG enrichment results of DEGs between these two groups ( Q value <0.05). (D) Spearman's correlation between SLC12A5 and predicted IC50 of drugs. Positive correlation means drug sensitive in SLC12A5 low‐expression group. (E) Targeted genes and pathways of drug listed in Figure .

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Mutagenesis, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: Construction of SLC12A5 overexpression cell lines. (A) Relative expression of SLC12A5 in clinical surgical samples by qPCR. Normal brain tissue: n = 3; Grade 2 glioma tissue: n = 3; Grade 3 glioma tissue: n = 3; Grade 4 glioma tissue: n = 3. (B) Relative expression of SLC12A5 in different cell lines by qPCR. (C) Expression level of SLC12A5 in GSE15824 data sets. Normal human astrocytes: NHA; Glioblastoma cell lines: LN018, LN215, LN229, LN319 and BS149. (D) Relative expression of SLC12A5 in LN229 and U87 cell lines under OE‐KCC2, OE‐NC and WT conditions by qPCR. OE‐KCC2: SLC12A5 overexpression; OE‐NC: negative control; WT: wild type. (E) Left: representative images of LN229 and U87 cell lines under OE‐KCC2 and OE‐NC conditions labelled by DAPI (Blue) and KCC2 (Red). Right: quantification of KCC2 intensity in different conditions.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Over Expression, Expressing, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: Overexpression of SLC12A5 suppresses glioma cell growth. (A) Left: Representative images of LN229 and U87 cell lines under OE‐KCC2 and OE‐NC conditions labelled by DAPI (Blue) and Ki67 (Red). Right: Quantification of Ki67‐positive cell number in different conditions. (B) Left: Wound healing area of LN229 and U87 cell lines under OE‐KCC2 and OE‐NC conditions. Right: Quantification of wound healing area in different conditions.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: GABA A receptor and synaptic activity may be involved in SLC12A5‐induced inhibition mechanisms. (A) Pearson coefficients between SCL12A5 and each GABA A channel subunits in TCGA glioma cohorts. * p < 0.05 and ** p < 0.01. (B) Correlation between SLC12A5 and average expression of GABA A receptor based on 19 subunit‐related genes in TCGA glioma cohorts. (C) Survival analysis in TCGA glioma cohorts by combining the expression of GABA A receptor and SLC12A5. (D) Four cellular states of tumour cell in single cell level based on related meta‐module score. (E) Different subtypes preference among SLC12A5‐positive and SLC12A5‐negative tumour cells. (F) The expression level of SLC12A5, GABA A receptor and synaptic genes in single‐cell RNA‐seq. Red points mean cells with the count of SLC12A5 exceeding 1; blue points mean cells with the enrichment score of synaptic genes exceeding 90% quantiles of it in NPC‐like cells; green points mean cells with the enrichment score of GABA A receptor exceeding 90% quantiles of it in NPC‐like cells; merge image is the superimposition of three colours. (G) Comparison of the enrichment score of GABA A receptor and synaptic genes between SLC12A5‐positive and SLC12A5‐negative tumour cells.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Activity Assay, Inhibition, Expressing, RNA Sequencing, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: A potassium‐chloride co‐transporter with altered genome architecture functions as a suppressor in glioma

doi: 10.1111/jcmm.18352

Figure Lengend Snippet: SLC12A5 contributes to tumour immune microenvironment and immunotherapy response. (A) eat map shows the correlation between SLC12A5 and tumour microenvironment features in TCGA glioma cohorts. CL, classical; ME, mesenchymal; NA, not applicable; NE, neural; PN, proneural; WT, wild type. (B) Correlation between SLC12A5 and immune score, stromal score and tumour purity calculated by ESTIMATE in TCGA glioma cohorts. (C) Comparison of anti/pro‐tumour immune infiltrate ratio and TGF‐beta pathway activity between SLC12A5 high‐expression group and low‐expression group in TCGA glioma cohorts. Anti‐tumour immune infiltrate: Average enrichment score of anti‐tumour immune signatures; Pro‐tumour immune infiltrate: Average enrichment score of anti‐tumour immune signatures. (D) Heat map shows the correlation between SLC12A5 and immune evasion signatures in TCGA glioma cohorts. (E) Survival analysis in a GBM cohorts accepted PD‐1 inhibitors treatment by combining the expression of PD‐L1 and SLC12A5. (F) Graphs show response efficacy of immunotherapy in four groups of GBM patients. NR, non‐responder; R, responder.

Article Snippet: SLC12A5 overexpression lentivirus (OE‐KCC2) was designed and synthesized by GENECHEM.

Techniques: Comparison, Activity Assay, Expressing