Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Altered astrocytic and microglial homeostasis characterizes a decreased proinflammatory state in bipolar disorder

doi: 10.1101/2023.10.29.564621

Figure Lengend Snippet: (A) Volcano plot of the astrocytic DEGs. (B) Pathways significantly modulated by the differentially expressed genes (DEGs) in astrocytes. (C) Result of the normalized quantification of [ 18 F]FDG γ counting from cells infected with either LV-mCherry or LV-ADCY2 viruses. Paired t-test, p = 0.0188. (D) Representing images of the NEUN (green), GFAP (grey) and ADCY2 (orange) immunofluorescence staining in the Cg of BD and CT subjects. Scale bar = 20 µm. (E) Results of the quantification of the mean grey value of ADCY2 in various cell types. Results of a three-way anova with effect on diagnosis (p = 0.02) and region (p = 0.0107), but no interaction effect between those 2 parameters (p = 0.9059). (F) Results of the quantification of the mean grey value of ADCY2 in NEUN + cells. Results of a three-way anova with effect only on diagnosis (p = 0.0282), but not on region (p = 0.3973). (G) Results of the quantification of the mean grey value of ADCY2 in GFAP + cells. Results of a three-way anova with effect on diagnosis (p = 0.02503) and region (p = 0.00514), but no interaction effect between the 2 parameters (p = 0.7985). (H) Results of the quantification of the mean grey value of ADCY2 in GFAP - /NEUN - cells. Results of a three-way anova with effect on diagnosis (p = 0.0278) and region (p < 0.005), but no interaction effect between the 2 parameters (p = 0.9289). (I) Representative image of NCAN immunohistochemistry staining in the Cg of CT (up) and BD (down) subjects. Scale bar = 250 µm. (J) Results of the quantification of NCAN-positive pixels over total pixel (in %) in the Cg of BD as compared with control (significance as the result of unilateral t-test, *: p < 0.05).

Article Snippet: Primary antibodies were used as follows: ADCY2 (orb34050, Biorbyt – 1/20), GFAP (MA5-12023, Invitrogen – 1/250), NEUN (266 004, Synaptic Systems – 1/250).

Techniques: Infection, Immunofluorescence, Staining, Biomarker Discovery, Immunohistochemistry, Control

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32β is the most dominant isoform in HepG2+LX-2 spheroids that increases neutral fat content, and IL32 downregulation lowers neutral fat in 2D cultured hepatocytes (A) To test the effect of IL-32 administration on intracellular fat content, immortalized human hepatic cell lines HepG2 and HepaRG were cultured in 2D and incubated with human recombinant IL-32α, IL-32β or IL-32γ isoform for 48 h. Then, intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ, showed increased intracellular neutral fat content in both (A) HepG2 (top) and HepaRG (bottom). (B) To test the effect of IL32 downregulation on intracellular fat content, 24 h after seeding, cells were transfected with scramble or IL32 siRNA and grown in regular medium without FBS (HepG2) or medium supplemented with 25 μM oleic acid (HepaRG) for an additional 48 h. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method. Intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ showed lower intracellular neutral fat content in both HepG2 (left) and HepaRG (right). Two-sided p values were calculated by the Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. (C) HepG2+LX-2 cells were cultured as spheroids for 96 h exposing them to medium supplemented with 1% BSA or to increasing concentrations of a mixture of fatty acids (PA + OA, 1:2). IL32 gene expression measured after exposure, demonstrated higher IL32 mRNA levels with increasing intra-spheroidal triglyceride levels. The p value was calculated by test for linear trend. Data shown as mean ± SD for the reported number of experiments. (D) Percentage of IL32 gene products versus other IL-32 isoforms was measured from RNA sequencing (RNA-seq) data and IL-32β is the most dominantly expressed IL32 isoform in our spheroid model. The p value was calculated by one-way ANOVA. OA, oleic acid; PA, palmitic acid; RU, relative unit.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Recombinant, Staining, Transfection, Knockdown, MANN-WHITNEY, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Incubation with human recombinant IL-32β increases while IL32 downregulation lowers intracellular triglyceride content in spheroids from immortalized and human primary hepatocytes (A) HepG2+LX-2 cells were cultured as spheroids for a total of 96 h. Initially, 48 h after seeding cells the media was supplemented with 25 nM IL-32β for 48 h. (B) HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation for total of 96 h. (C) Primary human hepatocytes (PHH) were cultured as spheroids for a total of 7 days. Initially, 48 h after seeding the media was supplemented with 25 nM IL-32β for an additional 5 days with media replacement every 48 h. (D) PHH were cultured as spheroids along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation, for a total of 7 days. For both spheroid models, cellular ATP levels (marker of viability) remained stable between the experimental groups. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method, relative to beta-actin. Intracellular neutral fat content measured by Oil Red O staining and AdipoRed assay showed an increase in triglycerides content after incubation with IL-32β while IL32 downregulation lowers triglyceride levels. Two-sided p values were calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. RFU, relative fluorescence unit; RU, relative unit (to beta-actin); SCR, scramble siRNA; untr, untreated.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Cell Culture, Generated, Negative Control, Transfection, Marker, Knockdown, Staining, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL32 downregulation lowers intracellular COL1A1, increases MMP2 levels, and lowers TIMP2 in primary di-lineage human spheroids Primary human hepatocytes and primary hepatic stellate cells, at the ratio 24:1, were seeded with negative control scramble (SCR) and IL32 siRNA, at 5,000 cells/well in ultra-low attachment 96-well U-bottom ultra-low attachment plates. Fifty percent of the total media was replenished with fresh media every 48 h. (A) After 7 days of formation, spheroids were collected and 8-μM sections were subjected to immunofluorescent staining for COL1A1. Immunofluorescence was quantified by ImageJ, normalized to number of DAPI stained nuclei. The knockdown efficiency was measured by real-time qPCR, relative to beta-actin. (B) MMP2, TIMP-1, TIMP-2, and α-SMA protein levels were measured by western blotting in the cell lysate. Calnexin was used as loading control. Representative images of protein levels are shown. For each panel, data shown as mean ± SD of the reported independent experiments. Two-sided p values were calculated by Mann-Whitney non-parametric test. COL1A1, collagen Iα1; MMP2, matrix metallopeptidase 2; TIMP1, tissue inhibitor of metalloproteinase 1; TIMP2, tissue inhibitor of metalloproteinase 2.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Negative Control, Staining, Immunofluorescence, Knockdown, Western Blot, Control, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Endogenous IL32 downregulation lowers, and incubation with recombinant IL-32 β increases, intracellular triglycerides synthesis For endogenous IL32 downregulation experiments, HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or IL32 siRNA transfection mix for a total of 96 h. For IL-32β incubation experiments, initially, 48 h after seeding HepG2+LX2 cells (24:1), the media was supplemented with 25 nM IL-32β for another 48 h. In both conditions, newly synthesized triglycerides were separated by TLC and quantified by scintillation counting after incubation with 6 μCi/mL 3 H-glycerol plus 1.5 mM glycerol for 12 h. (A) Reduction in de novo triglyceride synthesis after IL-32 downregulation. (D) Increase in de novo triglyceride synthesis after 25 nM IL-32β incubation. Cells were incubated with 8.5 μCi/mL 3 H-palmitate +55 μM/L palmitic acid for 6 h, after which palmitate was precipitated with BSA and perchloric acid and quantified by scintillation counting. (B and E) Graph shows no difference in beta oxidation in both experimental groups. APOB-100 synthesis and secretion levels were measured by immunoblotting. (C) Decrease in APOB-100 in cell lysate and cell culture supernatant after IL-32 downregulation. (F) No changes in APOB-100 levels in cell lysate and culture medium, after incubation with 25 nM IL-32β. The reported number of experiments were performed independently. Representative blots are presented. For all experiments, two-sided p value was calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD. AU, arbitrary units; DPM, disintegrations per minute; TAG, triacylglycerol.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Generated, Negative Control, Transfection, Synthesized, Western Blot, Cell Culture, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Differentially expressed genes reveals downregulation of key genes for lipid metabolism in HepG2+LX2 spheroids (A) Key genes of lipid metabolism differentially expressed in spheroids after IL32 downregulation as compared with scramble. (B) Top 100 differentially expressed genes after IL32 downregulation as compared with scramble. Data are presented as log2-fold change in expression and –log 10 of p values adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). VLDL, very low-density lipoprotein.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Co-downregulation of PLA2G2A and IL32 abolished the IL-32-mediated intracellular triglycerides lowering and IL32 downregulation reduces intracellular PI levels in human primary spheroids Primary human hepatocytes were cultured as spheroids and incubated with negative control (SCR) siRNA or 30 nM IL32 , and combination of IL32 and PLA2G2A for a total of 7 days. (A) Cellular ATP levels (marker of viability) were not different within the three groups. There was an 80%^–90% reduction in mRNA levels of IL32 and PLA2G2A , relative to beta-actin. Intracellular neutral lipid content (measured by Oil Red O staining) normalized to nuclei (stained by DAPI) were lower after IL32 downregulation while the co-downregulation of PLA2G2A and IL32 abolished this reduction. (B) IL32 downregulation results in higher PLA2G2A mRNA levels measured by real-time PCR. (C) IL32 and PLA2G2A were downregulated individually or in combination in primary hepatocyte spheroids and PLA2G2A levels were measured by human PLA2G2A ELISA in the culture medium. PLA2G2A levels were higher after IL32 downregulation and lower after co-downregulation of IL32 and PLA2G2A . (D) After 2 days from seeding, hepatocyte spheroids were incubated with 10, 25, and 50 nM human recombinant IL-32β for 5 days. PLA2G2A was measured from cell culture supernatant using human PLA2G2A ELISA and we observed a dose dependent decrease in secreted PLA2G2A levels with increasing concentration of IL-32β. The p values were calculated by test for linear trend. (E) Lipid fingerprint measured by liquid chromatography- quadrupole time-of-flight-mass spectrometry demonstrated lower total PI and triglycerides (top) levels after IL32 downregulation. There was a reduction in all PI species, except 40:5 and 40:6, with the largest effect size in 38:4 (bottom). For each part, data are shown as mean ± SD of the reported independent experiments. Two-sided p values calculated with unpaired t test for n = 3 and Mann-Whitney non-parametric t test for n > 3. Cer, ceramides; CL, cardiolipins; DAG, di-acylglycerides; GalCer, galactosyl ceramides; LPC, lysophosphatidylcholine; ORO, Oil Red O; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; RFU, relative fluorescence units; RU, relative units; PC, phosphatidylcholine; SM, sphingomyelin; TAG, triacylglycerols.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Negative Control, Marker, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Liquid Chromatography, Mass Spectrometry, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32 rs76580947 minor allele associates with lower IL-32 expression, severe liver steatosis, and lower liver non-invasive tests (A) Regional plots of association between IL32 common genetic variants (minor allele frequency of >0.01) and ALT in the European subset of UK biobank. The x axis shows the variant positions (GRCh37); the y axis shows the –log10 p values. The gray diamond represents rs76580947, with the strongest association in the plotted region (IL-32 ± 50 Kbp), for which its pairwise LD with other variants is color coded as shown on the figure. (B) The association between rs76580947 and hepatic IL-32 mRNA levels was tested in 207 individuals from the MAFALDA cohort adjusting for age, gender, percentage of coding bases (a quality control measure from the Picard toolkit), RNA Integrity Number (RIN), and five surrogate variables detected by surrogate variable analysis. Carriers of the variant have lower IL-32 mRNA levels. Data shown as violin plots and adjusted p values are reported. (C) The association between IL32 rs76580947 stratified by genotype and IL-32 plasma protein level in 365,495 European participants from UK Biobank was tested using a linear regression analysis adjusted for age, gender, body mass index, first 10 genomic principal components, and array batch. Violin plot shows the normalized Protein eXpression (NPX) values that were rank-based inverse normal transformed prior to the analysis. (D) Forest plot of association and meta-analysis for IL32 rs76580947 with steatosis in three independent cohorts: Southern Italy (N = 425), Central Italy (N = 245), and Finnish (N = 745). The plot shows protection against severe liver steatosis (steatosis absence or mild vs. severe; fixed-effect p = 0.027). The association was tested by a binary logistic regression analysis under an additive genetic model adjusted by age, gender, body mass index, and recruitment center (only for the Finnish cohort). Pooled effect estimates were calculated using inverse-variance-weighted fixed effects meta-analysis. (E) The association between IL32 rs76580947 and clinical liver fibrosis scores and APOB levels in 365,495 European participants from UK Biobank. The analysis was performed under an additive model, using linear regression adjusting for age, gender, body mass index, the first 10 genomic principal components, and array batch. All traits were rank-based inverse normal transformed prior to the analysis. CI, confidence interval.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing, Variant Assay, Control, Clinical Proteomics, Transformation Assay

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet:

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Recombinant, Protein Extraction, Staining, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Transfection, Gene Expression, Fluorescence, Software, Cell Counting

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b suppressed RANKL‐induced osteoclast differentiation without inducing cytotoxicity. (a) RAW264.7 cells were seeded onto 96‐well plates and incubated for 24 or 72 hr with different concentrations of IL‐37b. The CCK‐8 assay was used to detect cell proliferation. (b) Precursor cells were cultured with various concentrations of IL‐37b followed by M‐CSF (50 ng/ml) and RANKL (50 ng/ml) stimulation for 72 hr. These cells were simultaneously exposed to all of these factors. The steps for TRAP staining are described in the methods. Scale bar = 200 μm. (c) TRAP‐positive multinucleated cells ( ≥ 3 nuclei) were identified as osteoclasts and were counted. (d) BMMs were treated with M‐CSF (50 ng/ml), RANKL (50 ng/ml), and IL‐37 (200 ng/ml) for 5 days. (e) RAW264.7 cells were plated on the Osteo Assay Surface and were cultured with RANKL and M‐CSF for 6 days in the presence or absence of 200 ng/ml IL‐37b. Scale bar = 200 μm. Quantification of the bone resorption area on the Osteo Assay Surface. N = 4. The data are presented as the means ± SD. * p < 0.05, ** p < 0.01. CCK‐8: cell counting kit‐8; IL‐37b: interleukin‐37b; M‐CSF: macrophage colony stimulating factor; RANKL: receptor activator of nuclear factor‐κB ligand; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, CCK-8 Assay, Cell Culture, Staining, Cell Counting

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b inhibited the expression of specific mRNAs and proteins at different stages of osteoclast differentiation. The MyD88 dimerization inhibitor (ST 2825) reversed the effects of IL‐37b on osteoclastogenesis. (a) Cells were treated with RANKL (50 ng/ml), M‐CSF (50 ng/ml), and IL‐37b (200 ng/ml) for 24 or 72 hr, and then lysed for Western blot analyses using antibodies against NFATc1 and GAPDH. (b,c) The expression of genes associated with osteoclast differentiation, fusion, and function were detected by qRT‐PCR. * p < 0.05 and ** p < 0.01 compared with the RANKL‐treated control. (d) TRAP staining showed that 10 μg/ml ST 2825 partially counteracted the inhibitory effects of IL‐37b on osteoclast formation. Scale bar = 100 μm. (e) Western blot showing the level of NFATc1 in cells stimulated with RANKL in the presence or absence of ST 2825. The results were normalized to GAPDH levels. (f) Co‐IP analysis of the interaction between SIGIRR and MyD88 in RAW264.7 cells treated with RANKL and IL‐37b. Co‐IP: coimmunoprecipitation; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IL‐37b: Interleukin‐37b; mRNA: messenger RNA; MyD88: myeloid differentiation factor 88; M‐CSF: macrophage colony stimulating factor; NFATc1: nuclear factor of activated T cells 1; qRT‐PCR: quantitative real‐time polymerase chain reaction; RANKL: receptor activator of nuclear factor‐κB ligand; SIGIRR: single Ig IL‐1‐related receptor; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Staining, Co-Immunoprecipitation Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b decreased the phosphorylation of p65 and IκB, intermediates in the classical NF‐κB pathway, in cells stimulated with RANKL, thereby reducing the translocation of the NF‐κB p65 subunit into the nucleus. (a) RAW264.7 cells were plated in six‐well plates and pretreated with or without IL‐37b (200 ng/ml) for 2 hr in the presence of M‐CSF (50 ng/ml) before RANKL stimulation for 0, 5, 15, and 30 min. Whole‐cell lysates were subjected to Western blot analyses with the indicated antibodies. GAPDH was used as the internal control. (b) RAW264.7 cells were seeded in 96‐well plates and treated with IL‐37b (200 ng/ml) for 2 hr, followed by stimulation with 50 ng/mll RANKL for 30 min. The intracellular location of the p65 subunit was observed by immunofluorescence staining. Five cells were selected from the RANKL group and the IL‐37b intervention group, respectively. The gray values of the red and blue staining were measured using the ImageJ software, and the mean values were plotted using excel. Scale bar = 200 μm. DAPI: 4’,6‐diamidino‐2‐phenylindole; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IL‐37b: interleukin‐37b; M‐CSF: macrophage colony stimulating factor; NF‐κB: nuclear factor‐κB; RANKL: receptor activator of nuclear factor‐κB ligand [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Control, Immunofluorescence, Staining, Software

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: RAW264.7 cells that had been pretreated with RANKL for 24 hr were stimulated with LPS (100 ng/ml) to induce their differentiation into osteoclasts; IL‐37b reversed the process. (a) The CCK‐8 assay was used to examine the effect of LPS on progenitor cell proliferation. (b) IL‐37b inhibits RANKL or LPS‐mediated osteoclast fusion. A 24‐hr RANKL pretreatment triggered the fusion of precursor cells exposed to LPS conditions. Immunofluorescence staining revealed that IL‐37b reduced LPS‐induced actin ring formation. Scale bar = 200 μm. (c) After culture under the same induction conditions described above for three days, TRAP‐positive multinucleated cells were identified as osteoclasts. Scale bar = 200 μm. (d) RAW264.7 cells were treated with RANKL (50 ng/ml) for 24 hr and then stimulated with LPS (100 ng/ml) or LPS (100 ng/ml) and IL‐37b (200 ng/ml) for 24 hr. M‐CSF (50 ng/ml) was maintained in the culture media throughout the induction period. The relative expression levels of NFATc1 and c‐Fos were detected by quantitative PCR. (e) IL‐37b reduces the expression of proinflammatory cytokines during osteoclast differentiation. The levels of the IL‐1β, IL‐6, and TNF‐α mRNAs during the course of RANKL‐ or LPS‐induced osteoclastogenesis were analyzed by qRT‐PCR. *p < 0.05; ** p < 0.01 compared with the LPS‐treated group. CCK‐8: cell counting kit‐8; IL‐37b: interleukin‐37b; LPS: lipopolysaccharide; M‐CSF: macrophage colony stimulating factor; NFATc1: nuclear factor of activated T cells 1; qRT‐PCR: quantitative real‐time polymerase chain reaction; RANKL: receptor activator of nuclear factor‐κB ligand; TNF: tumor necrosis factor; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Recombinant human IL‐37b/IL‐1F7b protein (7585‐IL‐025/CF), recombinant murine RANKL (462‐TEC‐010/CF), and macrophage colony stimulating factor (M‐CSF; 416‐ML‐010) were from R&D Systems (Minneapolis, MN).

Techniques: CCK-8 Assay, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Counting

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 1. SMOC2 is highly upregulated in mice and humans with kidney fibrosis. Quantitative immunostaining for SMOC2 (red) and αSMA (green) was performed on kidney sections obtained from mice at day 7 following (A) unilateral ureteral obstruction (UUO) or (B) folic acid injection (FA) (n = 5; 20× magnification). Scale bar: 50 μm. For the UUO model, contralateral kidney (CoK) tissue from day 14 was also included. Bottom panel in A shows magnified images taken at 40× from the selected regions of the middle row. Scale bar: 30 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence, as represented in a box plot, was performed using representative images of 5 visual fields for each tissue analyzed. (C and D) Representative Western blot (n = 5/condition; Supplemental Figure 1, B and C [UUO and FA, respectively]) of SMOC2, αSMA, collagen 1α1, and fibronectin expression using kidney samples obtained from mice subjected to 7 and 14 days of UUO or FA. (E) Quantitative immunostaining for SMOC2 (red) and αSMA (green) in human kidneys with pathological fibrosis underlying chronic kidney disease (CKD) (n = 5) and nonfibrotic patients (n = 5; 20× magnification). Yellow scale bars: 50 μm. Bottom panel shows magnified images taken at 60× from the selected regions of the middle row. White scale bars: 25 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence as represented in a box plot was performed using representative images of 5 visual fields for each tissue analyzed. (F) Urinary levels of SMOC2 and kidney injury molecule-1 (KIM-1) normalized to urinary creatinine were measured in patients with CKD (n = 13) compared with healthy volunteers (n = 13). Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test. Yellow arrows, tubules. White arrows, interstitium.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Immunostaining, Injection, Quantitation Assay, Immunofluorescence, Western Blot, Expressing

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 2. SMOC2-overexpressing mice are more susceptible to kidney fibrosis than WT mice. (A) Confirmation of SMOC2 overexpression in SMOC2 transgenic (SMOC2 Tg) mice by PCR (above, primers specific to recognize Tg insert) and Western blotting (below) (Supplemental Figure 3A). (B) Repre- sentative Western blot (n = 5/condition; Supplemental Figure 3B and Supplemental Figure 5B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of unilateral ureteral obstruction (UUO). (C) Representative images of immunofluorescent staining for αSMA in CoK and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following UUO (n = 5/condition, 5 visual fields/ tissue). (D) Representative Western blot (n = 5/condition; Supplemental Figure 3C and Supplemental Figure 6B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of folic acid (FA). (E) Representative images of immunofluorescent staining for αSMA of normal and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following FA (n = 5/condition, 10 visual fields/ tissue). (F) Representative images of picrosirius red (n = 5/condition, 10 visual fields/tissue) and Masson’s trichrome (n = 5/condition, 5 visual fields/ tissue) staining of CoK versus 7 and 14 day UUO–treated kidneys. (G) Representative images of picrosirius red and Masson’s trichrome staining of normal versus 7 and 14 day FA–treated kidneys (n = 5/condition, 5 visual fields/tissue). Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Relative quantifications of images are represented as box plots, which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (CoK [UUO] or Normal [FA]) and #P < 0.05 (WT at respective time point) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Over Expression, Transgenic Assay, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 3. SMOC2 induces a fibroblast-to-myofibroblast transition. (A) RNAseq was performed using kidneys from SMOC2 Tg and WT mice at day 7 following UUO treatment. REVIGO treemap visualizations are shown for enriched gene ontology (GO) categories. Highly similar GO terms for cellular components are grouped and visualized by different colors and sizes of the rectangles using semantic similarity and enrichment P values. Representative Western blots of αSMA, collagen 1α1, and fibronectin from serum-deprived primary human kidney fibroblasts (B, n = 3/condition; Supplemental Figure 8C) and NIH3T3 fibroblasts (C, n = 3/condition; Supplemental Figure 8D) treated with 10 ng/ml SMOC2 with/without TGFβ1. (D) After 1 hour of antibody pretreatment, SMOC2 or TGFβ1 was treated to serum-deprived NIH3T3 cells for 24 hours and then tested for conventional fibrotic markers, while integrin β1 antibody was pretreated with NIH3T3 cells and then treated with SMOC2 (n = 3/condition; Supplemental Figure 8E). (E) NIH3T3 fibroblasts were transfected with SMOC2-MYC, empty vector control, or negative control MGP-MYC and then immunoprecipitated with a MYC (above) or integrin antibody (below). Western blots are representative immunoprecipitation experi- ments. (F) Representative Western blot for Phospho-Focal Adhesion Kinase (P-FAK) Y925, P-Myosin Light Chain (MLC) Ser19, and P-Paxillin Tyr118 from NIH3T3 cells treated with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 for 60 minutes (n = 5/condition; Supplemental Figure 8H). (G) Phalloidin staining of F-actin after NIH3T3 cells were treated 24 hours with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 (n = 3). Top row 40×, 25 μM. Bottom row 100×, 15 μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Staining

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 4. SMOC2 induces the properties of myofibroblast activities. (A) REVIGO treemap visualization for highly similar GO terms describing biological processes significantly different between SMOC2 Tg and WT mice at day 7 following UUO treatment. (B) Scratch assay performed on NIH3T3 cells treated 24 hours with 10 ng/ml SMOC2. Healing percentage represented in box plot (n = 5, 3 visual fields/condition; 10× magnification, scale bar: 50 μM). (C) Boyden chamber assay performed on NIH3T3 cells treated 24 hours with 10 ng/ml SMOC2. (D) NIH3T3 cells were treated 24 hours with/without 10 ng/ml SMOC2, and then trypsinized and reseeded. After 1 hour, unattached cells were washed and cell numbers were quantified for adherence (n = 3). (E) Meta- bolic activity of control and 10 ng/ml SMOC2-treated NIH3T3 cells were measured over time by MTT assay (n = 5). (F) NIH3T3 fibroblasts were treated 24 hours with/without 10 ng/ml SMOC2, and cell proliferation was assessed by EdU labeling and FACS (n = 5). Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Wound Healing Assay, Boyden Chamber Assay, Activity Assay, Control, MTT Assay, Labeling

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 5. Genetic inhibition of SMOC2 limits folic acid–induced kidney fibrosis in mice. (A) Confirmation of SMOC2 deletion in SMOC2-KO mice by PCR (above, PCR primers specific to recognize knock-in insert) and Western blotting (below). (B) Representative Western blot (n = 4/group; Supplemental Fig- ure 11) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to folic acid (FA) treatment. (C) Immunofluorescent αSMA staining of KO and WT kidneys at day 7 following with/out FA treatment (n = 4/group). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 from KO and WT mice. Confocal and light microscopy images are 20× magnification. Scale bars: 50μM. Quantification of images is represented as box plots (n = 4/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (WT normal) and #P <0.05 (WT at respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Inhibition, Knock-In, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 6. Genetic inhibition of SMOC2 limits UUO-induced kidney fibrosis in mice. (A) Representative Western blot (n = 5/group; Supplemental Figure 12) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to UUO. (B) Representative images (n = 3/group; 5 visual fields for each tissue analyzed) of immunofluorescent αSMA staining of KO and WT kidneys from normal mice and day 7 UUO mice. Relative quantitation is represented in a box plot as arbitrary units. (C) Masson’s trichrome staining of normal and 7-day UUO kidneys from WT and KO mice. Images of Masson’s trichrome staining are representative of 5–10 visual fields for each tissue analyzed. Quantification is represented in a box plot as arbitrary units (mice n = 5–6, 5–10 visual fields/mice). Confocal images are 20× magnification; scale bar: 50 μM. Light micros- copy images are 20× magnification; scale bar: 50μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and mini- mum and maximum values (bars). *P < 0.05 (WT CoK) and #P < 0.05 (WT at respective UUO) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Inhibition, Western Blot, Expressing, Staining, Quantitation Assay

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 7. Silencing SMOC2 reduces TGFβ1-induced fibrotic markers in vitro and folic acid–induced kidney fibrosis in mice. (A) Scheme of the exper- imental procedure for SMOC2 siRNA–transfected NIH3T3 cells. After 24 hours of treatment with SMOC2 siRNA or scrambled siRNA (ssiRNA), NIH3T3 fibroblasts were either treated with/without TGFβ1 for 24 hours. Representative Western blot (n = 3/condition; Supplemental Figure 14) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin expression. (B) Scheme of the experimental procedure for SMOC2 siRNA– or ssiRNA-injected C57BL/6 mice treated with/out folic acid (FA). Mice were injected i.v. with 30 μg/200 μl of SMOC2 siRNA or ssiRNA 4 hours before and 2, 4, and 6 days after an i.p. injection of 250 mg/kg of FA. Representative Western blot (n = 5/group; Supplemental Figure 16) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin. (C) Immunofluorescent αSMA staining of kidneys obtained from mice at day 7 following with/out FA either treated with ssiRNA or SMOC2 siRNA (n = 5). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 following ssiRNA or SMOC2 siRNA administration. Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Quantification of images is represented as a box plot (n = 5/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (ssiRNA + vehicle) and #P < 0.05 (ssiRNA respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: In Vitro, Transfection, Western Blot, Expressing, Injection, Staining, Light Microscopy

Journal: Mobile DNA

Article Title: An intronic LINE-1 regulates IFNAR1 expression in human immune cells.

doi: 10.1186/s13100-023-00308-3

Figure Lengend Snippet: Fig. 4 IFNAR1.L1M2a inducibly enhances transcription of IFNAR1 during type I IFN signaling. A IFNAR1 and three nearby genes, IFNAR2, IL10RB, and IFNGR2, were all considered to be possible targets of IFNAR1.L1M2a enhancer activity due to their proximity. B RNA sequencing of three wildtype clones (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) for IFNAR1.L1M2a-proximal genes during a 24 h time course of IFNβ treatment. Pairwise differential expression analysis [48] at the untreated timepoint showed slightly but significantly lower baseline expression of IFNAR1 and IFNGR2 in knockout cells, and likelihood ratio tests [48] showed significant differences in response to IFNβ treatment of IFNAR1 and IL10RB. At the 24 h timepoint, IFNAR1 expression was slightly but significantly upregulated compared to the untreated timepoint in wildtype cells but not in knockout cells, and expression of IFNAR1 was significantly lower in knockout cells. C-D Immunofluorescence was used to confirm protein-level differences in IFNAR1 expression between wildtype cells (left, gray) and knockout cells (right, purple) in untreated (top, light) and IFNβ-treated (bottom, dark) conditions. In agreement with RNA-seq data, IFNAR1 expression was induced by IFNβ treatment in wildtype cells, but not in knockout cells. Scale bar indicates 25 microns

Article Snippet: Cells were either untreated or were cultured in media containing 1000 U/ml human IFNɣ (#485-MI, R&D Systems) for 4 h before RNA extraction.

Techniques: Activity Assay, RNA Sequencing, Clone Assay, Knock-Out, Quantitative Proteomics, Expressing, Immunofluorescence

Journal: Mobile DNA

Article Title: An intronic LINE-1 regulates IFNAR1 expression in human immune cells.

doi: 10.1186/s13100-023-00308-3

Figure Lengend Snippet: Fig. 5 Deletion of IFNAR1.L1M2a.enh alters downstream transcriptional response to IFNβ. RNA sequencing of three wildtype (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) during a 24 h time course of IFNβ treatment. A Likelihood Ratio Tests [48] of RNAsequencing data revealed many genes with significantly altered IFNβ responses in knockout cells compared to wildtype. Many of these genes were expected IFN stimulated genes (red) as identified by induction in wildtype cells at the 4 h timepoint [48] (Additional file 7: Table S6). Gene ontology [51] confirmed that significantly differentially responsive genes were enriched for immune-related biological processes (red). B RNA sequencing of representative expected ISGs showed a dampened initial induction in the knockout cells (purple) compared to wildtype cells (gray) and differential expression throughout the time course of IFNβ treatment according to likelihood ratio tests [48]

Article Snippet: Cells were either untreated or were cultured in media containing 1000 U/ml human IFNɣ (#485-MI, R&D Systems) for 4 h before RNA extraction.

Techniques: RNA Sequencing, Knock-Out, Clone Assay, Quantitative Proteomics

Journal: British Journal of Pharmacology

Article Title: Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

doi: 10.1111/bph.14487

Figure Lengend Snippet: Cytotoxic effects of (A) melittin on HaCaT cells. Cell viability was determined by CCK‐8 assay ( n = 5). Effects of melittin on the chemokine expressions of cytokines (B) and chemokines ( n = 5) (C) in HaCaT cells that were stimulated by TNF‐α/IFN‐γ. T+I, TNF‐α/IFN‐γ‐stimulated; * P < 0.05 versus no treatment. + P < 0.05 versus TNF‐α/IFN‐γ alone.

Article Snippet: Recombinant human TNF‐α and IFN‐γ were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: CCK-8 Assay

Journal: British Journal of Pharmacology

Article Title: Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

doi: 10.1111/bph.14487

Figure Lengend Snippet: Effects of melittin on activation of the (A) JAK2, STAT1 and STAT3 signalling pathways in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (B) Immunofluorescence staining for p‐STAT1 and p‐STAT3 (labelled with Alexa Fluor 555, red), and F‐actin (labelled with Alexa Fluor 488, green). Cells were counterstained with Hoechst 33342 (blue). Effects of melittin on activation of the (C) NF‐κB signalling pathway in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (D) NF‐κB DNA binding activity in the nuclear extract was measured by EMSA. Representative images from each group. Scale bar = 50 μm. NC, normal control; T+I, TNF‐α/IFN‐γ‐stimulated; Mel1, TNF‐α/IFN‐γ‐stimulated +1 μg·mL −1 Mel.

Article Snippet: Recombinant human TNF‐α and IFN‐γ were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Binding Assay, Activity Assay, Control

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 modulates pancreatic tumor growth. A) Immortalized human pancreatic duct cells (hTERT-HPNE and HPDE6c7), human and murine PDA cell lines, and human patient derived-organoids (PDOs) demonstrate nSMase activity. Values shown are technical replicates of one lysate per cell line. B) SMPD3 expression was analyzed in an existing human pancreas organoid RNA-Seq dataset and is depicted in normal (n=11), primary (n=35), and metastatic (n=9) human organoids. Log of normalized counts of SMPD3 expression are plotted. C) Immunocytochemistry demonstrates nSMase2 expression in two pancreatic metastatic PDOs. Scale bar is 100uM. D) Nanoparticle tracking analysis of CD63 + sEVs upon GW4869 and vehicle treatment is depicted. E) IVIS imaging results at Day 7, 14, and 19 are graphed. Eight animals were injected with each cell line. F) Images depict bioluminescence signal during IVIS imaging at day 19. G) Pancreas weight/body weight at day 19 is graphed. Average values for the KPC scrambled shRNA are 0.04540 ± 0.001635 (n=8) and KPC Smpd3 shRNA 1 are 0.02817 ± 0.0008858 (n=7). H) Rag 1 KO mice injected orthotopically with KPC Smpd3 shRNA1 and KPC Smpd3 shRNA2 cell lines lived significantly longer than Rag1 KO mice injected orthotopically with KPC scrambled shRNA cells ( P =0.0013 using log-rank test and P =0.0001 using log-rank test, respectively). The median survival of Rag 1 KO mice injected orthotopically with KPC scrambled shRNA cells (n=12) is 19 days, KPC Smpd3 shRNA 1 cells (n=6) is 31.50 days, and KPC Smpd3 shRNA 2 cells (n=6) is 36.50 days.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Derivative Assay, Activity Assay, Expressing, RNA Sequencing, Immunocytochemistry, Imaging, Injection, shRNA

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: S m pd3 regulates proliferation of PDA cells in vivo . A) SMPD3 is expressed in a panel of human pancreatic duct cells (n=2 cell lines), human PDA cells (n=12 cell lines), and mouse PDA cells (n=3 cell lines). B) qPCR demonstrates a reduction in Smpd3 expression in KPC Smpd3 shRNA 1 LucFlag (n=3) and KPC Smpd3 shRNA 2 LucFlag (n=3) cell lines when compared to the KPC scrambled shRNA LucFlag (n=3) control cell line. C) Mean ± standard deviation for tumor volume of cell lines injected subcutaneously are graphed. Results of an unpaired t test comparing KPC scrambled shRNA and KPC Smpd3 shRNA 2 cell lines at day 17 are depicted on the graph. For all cell lines, N=6 animals. D) Mean ± standard error of the mean for tumor weight at Day 17 of cell lines injected subcutaneously are graphed. For all cell lines, N=6 animals. E) Immunofluorescence images and quantification of epithelial (CK19 + , Vimentin - , Dapi + ), proliferative (Ki67 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag line (n=4) are 39.33% ± 2.046%, KPC Smpd3 shRNA 1 LucFlag line (n=5) are 28.88% ± 4.096%, and KPC Smpd3 shRNA 2 LucFlag line (n=5) are 15.83% ± 1.960. Scale bar is 50uM. F) Immunofluorescence images and quantification of epithelial (E-cadherin + , Vimentin - , Dapi + ), apoptotic (Cleaved caspase 3 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.299% ± 0.2075%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.082% ± 0.2126%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=5) are 0.9832% ± 0.1307. Scale bar is 50uM. G) CTG assay or colony formation assay results for the indicated cell lines are depicted. H) Reduction of Smpd3 in epithelial pancreatic cancer cells modestly reduces fibrosis. IHC demonstrates loss of nSMase2 in epithelial pancreatic cancer cells in tumors generated using the KPC Smpd3 shRNA 1 LucFlag and KPC Smpd3 shRNA 2 LucFlag cell lines. One scale bar (1000uM) on the picrosirius red image is representative for all picrosirius red and corresponding brightfield images. One scale bar (200uM) on the nSMase2 IHC image is representative for all nSMase2 IHC images. I) Quantification of polarized light using the picrosirius red stained images is depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.734% ± 0.3760%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.319% ± 0.1328%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=4) are 0.9188% ± 0.1124.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: In Vivo, Expressing, shRNA, Control, Standard Deviation, Injection, Immunofluorescence, Generated, CTG Assay, Colony Assay, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 expression is upregulated in PanIN. A) Schematic details the construct design for the Smpd3 floxed mouse. B-C) For examination of nSMase2 expression in the indicated normal murine pancreatic cell types, nSMase2 IHC sections from 3 C57BL/6J mice were scored. Preneoplasia and neoplasia lesions were scored from KPC mice (n=10 animals scored for acinar-to-ductal metaplasia (ADM), n=12 animals scored for PanIN, n=10 animals scored for primary tumor, and n=4 animals scored for metastasis). Scale bars for IHC images are 40 uM. nSMase2 expression was scored on a scale of 0-3 and is depicted in (C). The scoring system used is 0=absent, 1=low, 2=medium, 3=high. Values depicted are representative for individual animals. D) Subcellular localization of nSMase2 was scored using the same slides and animals used for expression scoring in . Results from Chi-squared tests using a 2 x 3 table are shown. E-F) H&E and IHC for nSMase2 images are displayed for the indicated genotypes.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 ablation reduces formation of neoplasia and prolongs survival of KPC mice. A) KPC; Smpd3 f/f mice lived significantly longer than KPC; Smpd3 wt/wt mice ( P =0.0039 using log-rank (Mantel-Cox) test). The median survival of KPC; Smpd3 f/f mice (n=41) is 23.71 weeks, KPC; Smpd3 f/wt mice is 21.57 weeks (n=45), and KPC; Smpd3 wt/wt mice (n=47) is 20.00 weeks. B) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt mice (n=30), KPC; Smpd3 f/wt mice (n=27), and KPC; Smpd3 f/f mice (n=24). (C) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt (n=29), KPC; Smpd3 f/wt (n=25), and KPC; Smpd3 f/f (n=22) mice. Using Fisher’s exact tests, no significant differences were observed in the number or site of macrometastatic lesions present at end-stage in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , or KPC; Smpd3 f/f mice. If macrometastasis was present, only one site was observed per animal in these cohorts. D-E) Quantification of alcian blue staining demonstrates significantly reduced PanIN lesions in KPC; Smpd3 f/f mice (n=5) when compared to KPC; Smpd3 wt/wt mice (n=9) ( P =0.0154). H&Es used to score edema are depicted. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PanIN-bearing KPC; Smpd3 f/f mice. F) Edema scoring for KPC; Smpd3 wt/wt (n=9), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=7) mice is depicted. The scoring system used is 1=low, 2=medium, and 3=high. G-H) The percentage of pancreatic area occupied by PDA is significantly higher in KPC; Smpd3 wt/wt mice (74.16 ± 8.705) when compared to KPC; Smpd3 f/f mice (30.51 ± 17.72) ( P =0.0497) at 19-21 weeks of age. Average percent area is graphed in H (n=4 KPC; Smpd3 f/f mice, n=4 KPC; Smpd3 f/wt mice, n=5 KPC; Smpd3 wt/wt mice). Dotted lines show the PDA area in representative images. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PDA-bearing KPC; Smpd3 f/f mice at 19-21 weeks of age.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of the KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. A-F) Western blot depicts nSMase2 expression levels in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. G) Pictures of cell lines taken immediately before RNA isolation for RNA sequencing are shown. Scale bars are 100uM.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: RNA sequencing demonstrates a role for Smpd3 in modulation of cellular pathways and regulation of PDA subtype. A) Average luminescence values from 3-5 independent experiments per cell line are graphed. Average values for primary PDA cell lines generated from KPC; Smpd3 wt/wt mice (n=17 cell lines) are 98,614 ± 5,684, KPC; Smpd3 f/wt mice (n=14 cell lines) are 106,061 ± 6,699, and KPC; Smpd3 f/f mice (n=15 cell lines) are 88,460 ± 6,672. B) Average number of colonies from at least 3 independent colony formation assays per cell line are depicted. Average values from KPC; Smpd3 wt/wt mice (n=14 cell lines) are 26.02 ± 3.652, KPC; Smpd3 f/wt mice (n=14 cell lines) are 24.16 ± 2.519, and KPC; Smpd3 f/f mice (n=13 cell lines) are 21.34 ± 4.872. C) Heat maps depict expression of significantly deregulated molecules in Tumor Microenvironment Pathway and Hepatic Fibrosis Signaling Pathway in all three PPT cell lines. Molecules shown in heat maps include those molecules within the Ingenuity pathway analysis (IPA) pathway that were significantly deregulated in any comparison between PPT groups. D) IPA results show a bubble category chart of significantly deregulated pathways when comparing KPC; Smpd3 f/f PPT and KPC; Smpd3 wt/wt PPT cell lines. A positive z-score represents upregulation, and a negative z-score indicates downregulation of a pathway in KPC; Smpd3 f/f PPT when compared to KPC; Smpd3 wt/wt PPT cell lines. A gray circle depicts significant overrepresentation of a pathway, the direction of which cannot yet be determined. Select pathways are annotated. E) Picrosirius red stained images are shown for KPC; Smpd3 wt/wt (n=6), KPC; Smpd3 f/wt (n=5), and KPC; Smpd3 f/f (n=6) mice at 10-11 weeks of age. The small piece of intestine in the lower left corner of the KPC; Smpd3 f/wt image was excluded from the quantification. Scale bar is 1000uM. Inset shows brightfield image. F) Transcriptional subtyping of end-stage pancreatic tumor cell lines generated from KPC; Smpd3 wt/wt (PPT and MPT), KPC; Smpd3 f/wt (PPT), KPC; Smpd3 f/f (PPT and MPT) as well as KPC Mock OE and KPC Smpd3 OE lines is depicted. Genes used to identify subtypes according to the Moffitt classification are shown. G) Pie charts depict the number of cell lines with the classical and basal-like Moffitt subtypes. When comparing KPC; Smpd3 wt/wt PPT vs KPC; Smpd3 f/f PPT using Moffitt classification and Chi square test, p-value=0.0339.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Generated, Expressing, Comparison, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of RNA sequencing data from murine polyclonal pancreatic cancer cell lines. A) Heat map showing Integrin Signaling in all three PPT cell lines. B) Estimated counts for select genes identified from Sleuth comparison of polyclonal cell lines from end-stage PPTs of KPC; Smpd3 wt/wt (n=8), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=10) mice are graphed. Each box plot represents the distribution of estimated counts quantified by kallisto with 100 bootstrap samples. For each gene, the most significant protein-coding isoforms are shown. C) The top twenty pathways from IPA comparing KPC; Smpd3 f/f MPT vs KPC; Smpd3 wt/wt MPT are shown. D) A heat map including significantly differentially regulated molecules in the Pentose Phosphate Pathway are shown for KPC; Smpd3 wt/wt MPT and KPC; Smpd3 f/f MPT groups. E) The six differentially regulated pathways when comparing KPC Smpd3 OE vs KPC Mock OE are shown. F) Log of transcript per million (TPM) values for Smpd3 gene are graphed for KPC Mock OE and KPC Smpd3 OE sequenced cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: K P C ; Smpd3 f/f pancreata display significantly fewer activated stellate cells and fibroblasts when compared to both KPC; Smpd3 f/wt and KPC; Smpd3 wt/wt pancreata. A) Immunofluorescence images depict co-labeling with alpha SMA, Gfap, Desmin, and Dapi in the pancreas of 10–11-week-old mice. For KPC; Smpd3 wt/wt , n=6 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=5 animals. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel C. Scale bar is 20uM. B) Immunofluorescence images depict co-labeling with alpha SMA, Fap, Vimentin, and Dapi in the pancreas of 10–11-week-old mice. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel D. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, KPC; Smpd3 f/f , n=6 animals. Scale bar is 50uM. C) Quantification of alpha SMA, Gfap, Desmin, and Dapi quadruple positive cells is depicted. D) Quantification of alpha SMA, Fap, Vimentin, and Dapi quadruple positive cells is depicted. E) Pie charts depict Moffitt classification PDA subtypes for KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of lipidomics data from murine polyclonal pancreatic cancer cell lines. A) Heat map shows IPA Ceramide Signaling Pathway in all three PPT cell lines. B) Heat map shows differentially expressed lipids in comparisons amongst our murine polyclonal pancreatic cancer cell lines. Groups compared are listed as A-B where depicted upregulation or downregulation of a lipid would be in A when compared to B. C) Principal component analysis of our polyclonal murine pancreatic cancer cell lines based on lipid composition is depicted. D) Box plots showing expression of selected ceramide species in our KPC; Smpd3 wt/wt PPT, KPC; Smpd3 f/wt PPT, KPC; Smpd3 f/f PPT, KPC; Smpd3 wt/wt MPT, and KPC; Smpd3 f/f MPT cell lines. E) Box plots showing expression of selected phosphatidylcholine species in our KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of exosomes isolated from KPC; Smpd3 wt/wt PPT and KPC; Smpd3 f/f PPT cell lines. A) CD63eGFP particles per cell are graphed for KPC; Smpd3 wt/wt CD63eGFP cell lines (n=6) and KPC; Smpd3 f/f CD63eGFP cell lines (n=6). B) Fractions were subjected to WB for sEV markers to determine the fractions that exosomes are contained within using the C-DGUC exosome isolation protocol. Expected band sizes are listed for each marker. C) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt uninjected mice (n=30), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=15), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=15), KPC; Smpd3 f/f uninjected mice (n=24), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=9), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . D) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt uninjected mice (n=29), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=14), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=12), KPC; Smpd3 f/f uninjected mice (n=22), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=8), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . E) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 wt/wt are 0.04396 ± 0.006112 (n=28), KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.05477 ± 0.007678 (n=15), and KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 f/f are 0.06809 ± 0.009409 (n=16). F) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 f/f are 0.05085 ± 0.005912, KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.06149 ± 0.01434, and KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 f/f are 0.04654 ± 0.008279. G) Pancreas images at dissection for the indicated groups are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: PDA cell exosomes generated through nSMase2 accelerate PDA progression. A) Schematic of exosome injection study. B) Probability of survival for the indicated groups is graphed. Log-rank test was performed between the indicated groups. C-D) Volcano plot (C) and heat map (D) depict differentially expressed exosomal miRNAs isolated from KPC; Smpd3 f/f and KPC; Smpd3 wt/wt polyclonal PDA PPT cell lines. E) Exosomal protein abundance results for KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines are depicted. The x-axis shows the log2 fold change for KPC; Smpd3 f/f over KPC; Smpd3 wt/wt and the y-axis shows −log10(q-value). Significantly upregulated proteins (q-value < 0.05) are shown in green and significantly downregulated proteins are shown in red. F) Heatmap of top 40 differentially abundant exosomal proteins comparing KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt , displaying log2 normalized expression. Red indicates lower abundance of a given protein in each sample and green indicates higher abundance. Rows and columns are arranged based on hierarchical clustering dendrograms (not displayed). The color bar at the top indicates the genotype. G) IPA results depict significantly differentially regulated pathways when comparing exosomal proteins from KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Injection, Isolation, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: sEVs generated independently of nSMase2 promote a proinflammatory macrophage phenotype. A) Heat map depicts scaled marker expression in KPC; Smpd3 wt/wt and KPC Smpd3 f/f pancreata using our 17-marker myeloid spectral flow cytometry panel. B) t-SNE of 200,683 cells stained with our spectral flow panel depict 12 clusters identified for KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata through FlowSOM clustering. C) Heat map of scaled marker expression and number of cells for clusters shown in B is displayed. D) Bar graphs depict percent cellular subpopulations from KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata for the indicated markers after gating in FlowJo. E) Immunofluorescence images depict co-labeling with F4/80, iNOS, and Dapi at 10-11 weeks of age. Scale bar is 20uM. F) Quantification of intralobular F4/80 + and iNOS + copositive cells in KPC; Smpd3 wt/wt (n=5 animals), KPC; Smpd3 f/wt (n=6 animals), and KPC; Smpd3 f/f (n=5 animals) pancreata at 10-11 weeks of age is depicted. G) Schematic of experiment to assess how nSMase2-mediated exosome biogenesis affects polarization of macrophages. H-J) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Marker, Expressing, Flow Cytometry, Staining, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Functional effects of PDA cell sEVs on macrophages. A) Immunofluorescence images depict co-labeling with F4/80, Ly6G, and Dapi at 10-11 weeks of age. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=6 animals. Scale bar is 20uM. B) Quantification of interlobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. C) Quantification of intralobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. D-F) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: SMPD3 is an independent prognostic factor for pancreatic cancer patient survival. A) Log2 of the normalized counts are plotted for normal human pancreatic SMPD3 expression (n=39 samples) and primary PDA SMPD3 expression (n=39 samples). B) Log2 of the normalized counts are plotted for PPT SMPD3 expression (n=231 samples) and MPT SMPD3 expression (n=158 samples) in PDA patients. C) Kaplan-Meier survival curve of the whole cohort (N = 94) with the patients dichotomized into SMPD3 high and low expression based on the median mRNA expression value. SMPD3 high group had significantly better survival (median survival of 26.5 vs 15.0 months; P = 0.0364). A (0) value indicates a censored event and a (1) value indicates a death event. D) Kaplan-Meier survival curve of the subset of the whole cohort of patients who underwent adjuvant chemotherapy (> 95% single agent gemcitabine) after surgical resection (N = 58). SMPD3 expression was significantly associated with better survival (median survival of 34.3 vs 15.9 months; P = 0.0289) indicating SMPD3 is a biomarker of adjuvant gemcitabine response. E) Kaplan-Meier survival curve of the subset of the whole cohort of patients who did not undergo adjuvant chemotherapy after surgical resection (N = 36). SMPD3 expression was not associated with survival (P = 0.8001). F-G) Kaplan-Meier curves depict patient survival based on SMPD3 expression in locally advanced primaries or metastatic PDA from the COMPASS trial for patients who received chemotherapy with modified Folfirinox or Gemcitabine plus Abraxane. SMPD3 expression was stratified into high and low with low expression defined as the maximal chi-squared statistic. H) Log2 of SMPD3 expression by hypoxia status defined by . I) Log2 of SMPD3 expression in defined Waddell structural variation subtypes in COMPASS cases is depicted. J) Kaplan-Meier curves depict patient survival based on cytoplasmic and membranous nSMase2 expression in treatment naïve primary resected PDA. Tumor, and not stromal, cell nSMase2 expression was scored in 143 patients. When comparing survival of PDA patients with low cytoplasmic nSMase2 expression (n=89, mean survival=30.1 months) and high cytoplasmic nSMase2 expression (n=54, mean survival=29.1 months), p=0.627 using the log-rank test. When comparing survival of PDA patients with low membrane nSMase2 expression (n=65, mean survival=25.6 months) and high membrane nSMase2 expression (n=78, mean survival=32.9 months), p=0.052 using the log-rank test.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Biomarker Discovery, Modification, Membrane

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Pathways analysis of pancreatic nSMase2 expression in PDA patients based on treatment with chemotherapy in addition to the effect of nSMase2 expression on chemosensitivity and vasculature integrity. A) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs receiving adjuvant chemotherapy. The threshold line represents statistical significance. B) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs not receiving adjuvant chemotherapy. C) Representative images used to score the TMA for nSMase2 low and nSMase2 high membrane and cytoplasmic labeling. D) Images depict dextran and tomato lectin, a marker of blood vessels in mice, immunofluorescence at 10-11 weeks of age in KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreases. E) Percent pancreatic neoplastic area per field occupied by dextran is graphed for KPC; Smpd3 wt/wt (n=8) and KPC; Smpd3 f/f (n=6) mice. F) Negative log of gemcitabine IC50 for KPC; Smpd3 wt/wt , KPC; Smpd3 f/f , KPC Mock OE, and KPC Smpd3 OE cell lines is graphed. G) Quantification of total HIF1α in nSMase2 high and nSMase2 low primary pancreatic tumors of PDA patients is depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Membrane, Labeling, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 regulates PDA vasculature development. A-B) Heat maps showing VEGF Signaling and HIF1α Signaling pathways in all three PPT cell lines. C) Log of transcript per million (TPM) values for Vegfa gene are graphed for sequenced PPT cell lines. Wilcoxon test was performed. D) Pancreatic CD31 and CK19 immunolabeling at 19-21 weeks of age in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is depicted. Scale bar is 20uM. E) Quantification of pancreatic CD31 immunofluorescence in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is shown. F) Pancreatic CD31 and CK19 immunolabeling at 21 days post orthotopic injection of KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines into Rag1 KO mice 21 days post injection is depicted. Scale bar is 20uM. G) Quantification of pancreatic CD31 immunofluorescence in Rag1 KO mice injected with KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines 21 days post injection is depicted. H) Immunohistochemistry of CD31 in primary tumors from PDA patients is depicted with low and high nSMase2 expression. I) Percent CD31 positive area in primary nSMase2 low (n=19) and nSMase2 high (n=34) pancreatic tumors from PDA patients is shown. J) Immunohistochemistry of HIF1α is depicted in primary tumors from PDA patients with low and high nSMase2 expression. K) Percent nuclear HIF1α epithelial tumor cells in primary nSMase2 low (n=5) and nSMase2 high (n=6) PDA patient pancreatic tumors is shown. L) Ratio of pancreas weight to body weight for gemcitabine and vehicle treated Rag1 KO mice 21 days after pancreatic orthotopic injection of the depicted cell lines is shown. For KPC scrambled shRNA vehicle treated mice, n=4, for KPC scrambled shRNA gemcitabine treated mice, n=9, for KPC Smpd3 shRNA 1 vehicle treated mice, n=10, for KPC Smpd3 shRNA 1 gemcitabine treated mice, n=10, for KPC Smpd3 shRNA 2 vehicle treated mice, n=10, and for KPC Smpd3 shRNA 2 gemcitabine treated mice, n=10. M) Kaplan-Meier survival curves show probability of survival for vehicle-injected KPC; Smpd3 wt/wt mice (n=12, median survival=20.29 weeks), gemcitabine-injected KPC; Smpd3 wt/wt mice (n=19, median survival=23.43 weeks), vehicle-injected KPC; Smpd3 f/f mice (n=15, median survival=23.29 weeks), gemcitabine-injected KPC; Smpd3 f/f mice (n=16, median survival=19.86 weeks). A black line indicates a censored animal.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Protein-Protein interactions, Immunolabeling, Immunofluorescence, Injection, shRNA, Immunohistochemistry, Expressing