Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: RT‒qPCR primers.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Sequencing

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: Antibodies used for immunofluorescence staining.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Immunofluorescence, Staining

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: Antibodies used for western blotting.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Western Blot, Incubation

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Representative fluorescence microscopy images of the human fetal pancreas at week 13 (week 13) stained with antibodies against SPOCK2 (green) and C-peptide (C-PEP, red) to label early β-cells (upper panel). SPOCK2 (green) and glucagon (GCG, red) labeling of early α-cells (lower panel). Scale bars = 100 μm. DAPI (blue) was used to label the nuclei. N = 3 tissue samples. Examples of protein coexpression are presented in insets in the top corner of the merged images. b Representative confocal microscopy images of hPSC-derived β-cells (SC-β-cells) stained with antibodies against SPOCK2 (green), C-peptide (red) and PDX1 (white). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. An example of protein coexpression is presented in the inset in the top left corner of the merged image. c Western blot analysis of SPOCK2 protein levels in undifferentiated hPSCs (Day 0, D0) and SC-β-cells (Day 36, D36). An antibody against GAPDH was used as a loading control. Two bands correspond to nonglycosylated and glycosylated SPOCK2 protein. d Representative fluorescence microscopy images of the EndoC-βH1 cells stained with antibodies against SPOCK2 (green) and C-peptide (red). DAPI (blue) was used to label the nuclei. Examples of protein coexpression are presented as insets in the top corner of the merged images. Scale bars = 100 μm.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Fluorescence, Microscopy, Staining, Labeling, Confocal Microscopy, Derivative Assay, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Schematic outline of the bidirectional modulation of SPOCK2 expression. To knock down (KD) SPOCK2 expression, shRNAs or siRNAs against SPOCK2 were applied to EndoC-βH1 cells. To increase SPOCK2 expression, transient overexpression of the SPOCK2 transcript or recombinant SPOCK2 (rh SPOCK2) was used. To characterize EndoC-βH1 cells with the changes in SPOCK2 expression, live imaging, RNA-seq, IF or GSIS was used. b RT‒qPCR analysis of SPOCK2 mRNA levels in SPOCK2-KD, GIPZ-treated, and WT cells. One-way ANOVA for multiple comparisons was used to determine the p values. The data are presented as the means ± SDs. N = 3 biological replicates. c Representative fluorescence microscopy images of EndoC-βH1 SPOCK2-KD and GIPZ control cells stained with an antibody against SPOCK2 (green) showing lower levels of the SPOCK2 protein in SPOCK2-KD cells than in GIPZ control cells. Scale bars = 100 μm. DAPI (blue) was used to label the nuclei. d Western blot analysis of the SPOCK2 protein levels in EndoC-βH1 SPOCK2-KD and WT cells. An antibody against GAPDH was used as a loading control. e RT‒qPCR analysis of SPOCK2 mRNA in SPOCK2 OE and WT cells. The p values were assessed by an unpaired two-tailed Student’s t test. The data are presented as the means ± SDs. N = 3 biological replicates. f FACS analysis of SPOCK2 protein levels ( x -axis) in SPOCK2 OE (dark blue peak) and WT cells (dark green peak). SPOCK2 OE and WT cells incubated with secondary antibodies alone were used as controls, corresponding to the light blue and green peaks, respectively. g Higher SPOCK2 protein levels were detected in EndoC-βH1 SPOCK2 OE cells than in WT cells, as demonstrated by western blotting. An antibody against tubulin was used as a loading control. h EndoC-βH1 SPOCK2-KD, SPOCK2-OE, and WT cells were monitored via live cell imaging over a five-day culture period. Representative bright-field images (cells marked with a yellow mask) of EndoC-βH1 WT, SPOCK2-KD, and SPOCK2-OE cells on Days 0, 1, 3, and 5 are shown. Scale bars = 100 μm. i Quantification of phase object counts over a five-day time course normalized against the 12 h time point, showing the difference in total cell number between SPOCK2-KD (red) and SPOCK2-OE (blue) cells relative to WT cells (green). N = 3 biological replicates. j Representative fluorescence microscopy images of EndoC-βH1 WT (top), GIPZ control (middle), and SPOCK2 KD (bottom) cells stained with an antibody against the proliferation marker pHH3 (red). Examples of pHH3 nuclear detection with DAPI (blue) staining of the nuclei are presented as insets in the top right corners of the merged images. Scale bars = 100 μm. k Quantification of pHH3 + cells in EndoC-βH1 WT, GIPZ control, and SPOCK2 KD cells, shown as the ratio of pHH3 + to total live cells (DAPI + ). The larger number of SPOCK2-KD cells expressing pHH3 than WT and GIPZ control cells expressing this protein is shown as a %. One-way ANOVA for multiple comparisons was used to determine the p values shown on the graph. The data are presented as the means ± SDs. N = 9–12 biological replicates. l Spectral flow cytometry analysis of SPOCK2 and Ki67 protein expression in WT EndoC-βH1 cells, showing a population of proliferating cells with low SPOCK2 levels (SPOCK2-low/Ki67 + ) and a population of nonproliferating cells with higher SPOCK2 levels (SPOCK2-high/Ki67-). m Coexpression of Ki67 (red) and C-PEP (white) in SPOCK2-KD EndoC-βH1 cells. Scale bars = 100 μm. DAPI (blue) was used to stain the nuclei. Enlarged views are shown in the insets in the top corner of the images.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Expressing, Knockdown, Over Expression, Recombinant, Imaging, RNA Sequencing, Fluorescence, Microscopy, Control, Staining, Western Blot, Two Tailed Test, Incubation, Live Cell Imaging, Marker, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Schematic outline of the experimental design used to study the role of SPOCK2 in SC-β-cells. SPOCK2-KD, shRNAs against SPOCK2 or control shRNAs were added to the cells at the EP stage. In contrast, cells at the EP stage were treated with recombinant SPOCK2 (rh SPOCK2) protein. For SPOCK2 knockout (KO), CRISPR/Cas9 and clonal selection were used to delete the SPOCK2 gene in hPSCs, followed by differentiation into β-cells. Early hPSC-derived β-cells were subjected to proliferation analysis, IF, GSIS or scRNA-seq. DE: definitive endoderm, PP: pancreatic progenitors, EP: endocrine progenitors b Representative fluorescence microscopy images of cells during hPSC differentiation into β-cells stained with antibodies against markers of consecutive differentiation stages: definitive endoderm (DE) cells expressing SOX17 (green); pancreatic progenitors (PP) co expressing PDX1 (green) and NKX6-1 (red); endocrine progenitors (EP) stained for CHGA (green), SC-β-cells stained for C-PEP (green) and CHGA (red). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. c Representative fluorescence microscopy images of WT and SPOCK2-KD SC-β-cells stained with an antibody against SPOCK2 (red). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. d Quantification of normalized fluorescence signals from Fig. 3C. N = 5 biological replicates. e Quantification of proliferating SC-β-cells. The ratio of C-PEP + /pHH3 + cells to C-PEP + WT, GIPZ, or SPOCK2-KD cells. The larger number of SPOCK2-KD SC-β-cells expressing pHH3 than control cells is shown as a percentage. One-way ANOVA for multiple comparisons was used to determine the p values. The data are presented as the means ± SDs. N = 5 biological replicates. f SPOCK2-KO hPSC line generation. The top 3 sgRNAs targeting SPOCK2 exon 1 were cotransfected into hPSC-iCas9, and clones with frameshifts and introduction of the stop codon in exon 3 were selected for further analysis. Bottom: Agarose gel electrophoresis of DNA from selected single-cell clones of the SPOCK2-/- (KO) line compared with the WT line. g UMAP representation of single-cell transcriptomes of 5,500 SPOCK2 KO and 6,400 WT SC-β single cells grouped via graph-based clustering, with 7 distinct clusters: PP1—proliferating pancreatic progenitors; PP2—pancreatic progenitors; EP1—early endocrine progenitors; EP2—late endocrine progenitors; EC—enterochromaffin; MH—multihormonal cells; and β—immature β-cells. h Bubble plot of unique marker transcripts (shown on the x -axis) enriched in pancreatic progenitors (PPs), endocrine progenitors (EPs), or endocrine cell clusters (shown on the y -axis). The bubble size represents the percentage of cells expressing a gene, whereas the color intensity indicates the average scaled gene expression in a sample. i Cell cycle scoring of SPOCK2 KO and WT SC-β-cell scRNA-seq data showing the proportion (%) of SPOCK2 KO and WT SC-β-cells (defined as INS + /NKX6.1 + ) in phases G1, S and G2M. j UMAP plot showing the distribution of WT (left) and SPOCK2-KO (right) SC-β-cells in different phases of the cell cycle. The cells in phase G1 are red, those in G2M are green, and those in S are blue. k Violin plot showing INS expression levels in SPOCK2 KO and WT SC-β-cells in different cell cycle phases.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Control, Recombinant, Knock-Out, CRISPR, Selection, Derivative Assay, Fluorescence, Microscopy, Staining, Expressing, Clone Assay, Agarose Gel Electrophoresis, Marker, Gene Expression

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Bubble plot showing the relative expression of selected β-cell-associated genes in SPOCK2-KO and WT SC-β-cells. The bubble size represents the percentage of cells expressing a gene, whereas the color intensity indicates the average scaled gene expression in a sample. Pink signifies upregulation, and turquoise indicates downregulation of gene expression. b Representative confocal microscopy images of SPOCK2 KO and WT SC-β-cells stained with antibodies against C-PEP (green) and NKX6-1 (red). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. c Quantification of fluorescence signals from C-PEP and NKX6-1 staining (Fig. 4B). Higher protein levels were detected in SPOCK2-KO SC-β-cells than in WT SC-β-cells, with p values determined via Student’s t test and presented in the graph. The data represent the means ± SDs from 4 biological replicates. d Representative confocal microscopy images of SPOCK2 KO and WT SC-β-cells stained with antibodies against C-PEP (green) and PCSK1 (red). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. e Quantification of fluorescence signals from PCSK1-stained cells (Fig. 4D). Student’s t test was used to determine the p values shown on the graph. The data are presented as the means ± SDs. N = 5 biological replicates. f Insulin secretion from WT or SPOCK2-KD SC-β-cells and human primary islets challenged with low glucose (2.8 mM) or high glucose (16.7 mM) was normalized to the total cell number. One-way ANOVA for multiple comparisons was used to determine the p values shown on the graph. The data are presented as the means ± SDs. N = 4 biological replicates. g Insulin stimulation index of WT and SPOCK2-KD SC-β-cells compared with that of human primary islets, calculated as the ratio of insulin secretion in response to 16.7 mM vs. 2.8 mM glucose. One-way ANOVA for multiple comparisons was used to determine the p values shown on the graph. The data are presented as the means ± SDs. N = 4 biological replicates. h Electron microscopy images of WT and SPOCK2-KD EndoC-βH1 cells, with representative insulin granules shown in red; scale bar = 500 nm. i Box and whisker plot of the number of insulin granules per cell. The thick horizontal line indicates the median. N = 5 WT and SPOCK2-KD EndoC-βH1 cells.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Expressing, Gene Expression, Confocal Microscopy, Staining, Fluorescence, Electron Microscopy, Whisker Assay

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Schematic outline of the experimental design used to study the role of candidate genes in EndoC-βH1 and SC-β-cells. On the basis of the RNA-seq data from the SPOCK2-KD and SPOCK2-OE strains, MMP2 was selected as a candidate gene. EndoC-βH1 or SC-β-cells were treated with recombinant MMP2 (rh MMP2) and further subjected to live imaging, zymography, IF or GSIS. b Volcano plot of DEGs between EndoC-βH1 SPOCK2-KD and GIPZ control cells, with selected genes marked. The violet dots correspond to upregulated (-log10 (adjp) ≥2 and log2FC ≥ 0.5), and the yellow dots correspond to downregulated (-log10 (adjp) ≥2 and log2FC ≤ –0.05) genes. c Volcano plot depicting DEGs between EndoC-βH1 SPOCK2 OE and WT cells, with selected genes marked. The violet dots correspond to upregulated (-log10 (adjp) ≥2 and log2FC ≥ 0.5), and the yellow dots correspond to downregulated (-log10 (adjp) ≥2 and log2FC ≤ -0.05) genes. d RT‒qPCR results showing the expression of MMP2 and the MMP inhibitor TIMP1 . Student’s t test was used to determine significance. The data are presented as the means ± SDs. * ≤0.05. N = 3 biological replicates. e Zymography showing increased MMP2 activity in SPOCK2-KD EndoC-βH1 cells. f Representative fluorescence microscopy images of EndoC-βH1 WT cells treated for seven days with 15 ng/mL rh MMP2 protein and stained with an antibody against pHH3 (red). Untreated WT cells served as a control (none). DAPI (blue) was used to stain the nuclei. Scale bars = 100 μm. g Quantification of pHH3 + EndoC-βH1 WT cells, shown as the ratio of pHH3 + cells to the total number of DAPI + cells. The greater percentage of pHH3 + EndoC-βH1 after treatment with 15 ng/mL rh MMP2 is shown as a %. One-way ANOVA for multiple comparisons was used to determine the p value shown on the graph. The data are presented as the means ± SDs. N = 5 biological replicates. h Representative bright-field images of cells marked with a yellow mask and stained with an antibody against pHH3 (red). Compared with DMSO-treated cells, EndoC-βH1 WT cells treated with 5 μM MMP2i for five days presented fewer total cells (confluent) and pHH3 + cells. Scale bars = 100 μm. i Quantification of the confluence of EndoC-βH1 cells treated with different concentrations of MMP2i for five days normalized to 0 h. N = 3 biological replicates. j Quantification of proliferating SC-β-cells (INS + /Ki67 + ) via flow cytometry after MMP2 and CHIR99021 treatment; the results are presented as a percentage of the control values. One-way ANOVA for multiple comparisons was used to determine the p value shown on the graph. The data are presented as the means ± SDs. N = 3 biological replicates.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: RNA Sequencing, Recombinant, Imaging, Zymography, Control, Expressing, Activity Assay, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2

doi: 10.1038/s12276-024-01380-2

Figure Lengend Snippet: a Graphical representation of the top-ranking enriched ( p value ≤ 0.05) KEGG and Wiki pathways among DEGs in proliferating SPOCK2 KO SC-β-cells. b Bubble plot showing the relative expression of selected integrin signaling and JUN target genes in proliferating SPOCK2-KO and WT SC-β-cells. The bubble size represents the percentage of expressing cells, and the color intensity reflects average scaled gene expression (pink for upregulation, turquoise for downregulation). c Western blot analysis of phospho-FAK protein levels in WT or SPOCK2-KD EndoC-βH1 cells. An antibody against GAPDH was used as a loading control. d Differentially expressed AP1 targets identified via RNA-seq analysis of SPOCK2-KD vs. GIPZ and SPOCK2-OE vs. WT EndoC-βH1 cells. e Phospho-c-JUN protein levels in WT and SPOCK2-KD SC-β-cells, as determined by western blotting. An antibody against GADPH was used as a loading control. f Representative fluorescence microscopy images of EndoC-βH1 cells treated with CHIR, rh MMP2 or rhMMP2 together with CHIR and stained with an antibody against phospho-c-JUN (p-c-JUN red). Examples of p-c-JUN nuclear detection with DAPI (blue) costaining of the nuclei are presented in insets in the top right corners of the merged images. Scale bars = 100 μm. g Quantification of phospho-c-JUN + EndoC-βH1 cells treated with CHIR, MMP2 or MMP2 together with CHIR, shown as the ratio of cells expressing the protein to the total number of DAPI + cells. The larger number of EndoC-βH1 cells expressing phospho-c-JUN after MMP2 + CHIR treatment than WT cells expressing this protein are shown as a percentage. One-way ANOVA for multiple comparisons was used to determine the p values shown on the graph. The data are presented as the means ± SDs. N = 3 biological replicates. h Treatment with the JUN/JNK small molecule inhibitor SP600125 diminished MMP2-induced SC-β-cell proliferation. SC-β-cells were treated for 7 days with MMP2 (15 ng/ml), MMP2 + DMSO or MMP2 with 20 μM or 40 μM SP600125, after which the cells were stained for INS and pHH3 and quantitatively analyzed via flow cytometry. The data are presented as the means ± SDs. N = 6 biological replicates. One-way ANOVA for multiple comparisons was used to determine the p values shown on the graph. i Proposed molecular mechanism underlying SPOCK2-dependent β-cell proliferation and function. Reduced SPOCK2 expression or MMP2 treatment induces human β-cell proliferation via β-integrin-JUN pathway activation. In β-cells, SPOCK2 regulates MMP2, inhibiting its function. When SPOCK2 is knocked down, MMP2 cleaves ECM components, including collagen IV and fibronectin. This releases growth factors and ECM fragments, which act as substrates for the β-integrin receptor. Upon activation, the integrin receptor induces FAK autophosphorylation at Y397, activating downstream effectors, including c-JUN, leading to cell proliferation, the upregulation of β-cell markers (INS, PDX1, CHGA) and an increased number of insulin granules. Consequently, GSIS is improved in the short-term and long-term β-cells both in vitro and in vivo.

Article Snippet: The resulting SPOCK2 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB).

Techniques: Expressing, Gene Expression, Western Blot, Control, RNA Sequencing, Fluorescence, Microscopy, Staining, Flow Cytometry, Activation Assay, In Vitro, In Vivo

Journal: Diagnostics

Article Title: Identification and Validation of T-Cell Exhaustion Signature for Predicting Prognosis and Immune Response in Pancreatic Cancer by Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data

doi: 10.3390/diagnostics14060667

Figure Lengend Snippet: Expression validation of risk genes in PACA tissues. ( A ) The mRNA levels of risk genes in the TCGA-GTEx cohort. The differences were examined by Wilcoxon test and the box plot was drawn using the ggpubr (v0.6.0) R package. ( B ) The Kaplan–Meier curve of the high- and low-risk patients based on the expression of risk genes in TCGA cohort drawn using survival (v3.3.1) and survminer (v0.4.9) R packages. ( C ) The protein levels of SPOCK2, MT1X, and LIPH in PACA from the CPTAC database ( https://ualcan.path.uab.edu/index.html , accessed on 1 January 2024). ( D ) IHC staining of SPOCK2, MT1X, and LIPH in PACA tumor and adjacent tissues from untreated male PACA patients ( n = 3). **** represents p -value < 0.0001.

Article Snippet: The surgical PACA tumor and adjacent tissues were briefly deparaffinized and rehydrated, followed by incubation with primary antibodies overnight (anti-SPOCK2: 11725-1-AP; anti-MT1X: 7172-1-AP; anti-LIPH: 16602-1-AP) from Proteintech ( https://ptgcn.com/products/ , accessed on 1 January 2024).

Techniques: Expressing, Biomarker Discovery, Immunohistochemistry