Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a m 6 A MeRIP-seq indicates distribution of m 6 A peaks in different regions (5’UTR, first exon, other exon, and 3’UTR) of transcripts. Pie chart shows the percentage of m 6 A peaks within distinct regions of transcripts in LFS astrocytes. b Violin plot demonstrates significant elevation of highly m 6 A-marked transcripts upon YTHDF2 depletion. c Examination of YTHDF2 IP enrichment by eCLIP-seq. d Metagene plot of YTHDF2 eCLIP-seq indicates enrichment of YTHDF2-interacting mRNA peaks in the 3’UTR clustered around stop codons. e Motif analysis demonstrates that YTHDF2 binding motifs are similar to the consensus m 6 A motif RRACH. f Venn diagram identifying 84 YTHDF2-targeted m 6 A transcripts validated by a combination of m 6 A MeRIP-seq, YTHDF2 eCLIP-seq, and RNA-seq in LFS astrocytes. These transcripts include CDKN2B and SPOCK2 mRNAs. g IGV track views of m 6 A peaks located on CDKN2B and SPOCK2 transcripts in LFS astrocytes. h RT-qPCR indicates decreased expression of YTHDF2-targeted CDKN2B and SPOCK2 transcripts in LFS astrocytes ( n = 3 biologically independent samples). i Low expression of YTHDF2-targeted CDKN2B and SPOCK2 is correlated with poor overall survival of LGG/GBM patients. Log-rank (Mantel–Cox) test is performed to compute significance. j Immunostaining demonstrates lower CDKN2B in engrafted LFS cerebral organoids (upper panel) and increased CDKN2B in YTHDF2-depleted engrafted LFS cerebral organoids (lower panel), Scale bar, 100 µm. Anti-CDKN2B antibodies only recognize human but not mouse CDKN2B proteins. k mRNA stability assay demonstrates that YTHDF2 knockdown leads to an increased half-life of CDKN2B and SPOCK2 mRNAs. shCtrl-LFS and shYTHDF2-LFS astrocytes are treated with actinomycin D and total RNAs are isolated at 0, 30, and 60 min. ( n = 3 biologically independent samples). l RT-qPCR demonstrates elevated expression of YTHDF2 targets CDKN2B and SPOCK2 upon depletion of p53, YTHDF2, or SVIL as well as inhibition of MLL1 function by OICR-9429 or MI-2-2 in LFS astrocytes ( n = 3 biologically independent samples). The results are representative of at least three independent experiments ( c , j ). The data are presented as the mean ± SEM); two-way ANOVA with Bonferroni’s multiple comparison test ( l ); unpaired two-tailed Student’s t test ( h ); multiple t test ( k ); ** P < 0.01, *** P < 0.001. Source data and exact P values are provided in the Source Data file.

Article Snippet: Antibodies against p53 (1:1000, Santa Cruz, sc-126), SOX2 (1:200, Santa Cruz, sc-17320), SOX10 (1:200, Santa Cruz, sc-17342), OLIG2 (1:200, R&D Systems, BAF2418), GFAP (1:200, BioLegend, 837201), β-TUBULIN III (1:200, Sigma-Aldrich, SAB4200715), NESTIN (1:200, BioLegend, 655102), Ki67 (1:200, Life Technologies, 14-5698-82), VCL (1:2000, Sigma-Aldrich, V4505), PAX6 (1:200, Biolegend, 901301), Flag (1:2000, Sigma-Aldrich, F1804), HA (1:2000, Roche, 11666606001), V5 (1:2000, Thermo Fisher Scientific, R960-25), GFP (1:2000, Santa Cruz, sc-9996), m 6 A (1:1000, Synaptic Systems, 202003), YTHDF2 (1:1000, Proteintech, 24744-1-AP; 1:200, Aviva, ARP67917_P050), SVIL (1:500, Sigma-Aldrich, S8695), STEM121 (1:200, Takara Bio, Y40410), human nuclear antigen (1:200, Novus Biologicals, NBP2-34342), MLL1 (1:500, Bethyl Laboratories, A300-086A), H3K4me3 (1:200, Abcam, ab8580), H3K27Ac (1:200, Abcam, ab4729), METTL3 (1:1000, Proteintech, 15073-1-AP), METTL14 (1:1000, Cell Signaling, 48699), WTAP (1:1000, ABclonal Technology, A14695), FTO (1:1000, ABclonal Technology, A3851), ALKBH5 (1:1000, ABclonal Technology, A11684), CDKN2B (1:200, Thermo Fisher Scientific, MA1-12294), and SPOCK2 (1:200, Bioss Antibodies, BS-11966R) were purchased from the indicated suppliers.

Techniques: Binding Assay, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Immunostaining, Stability Assay, Knockdown, Isolation, Inhibition, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a In vivo mouse xenograft study demonstrates that knockdown of YTHDF2, SVIL, or MLL1 hampers LNZ308-p53(G245D) tumor growth ( n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. b Expression of CDKN2B or SPOCK2 inhibits cell proliferation of LFS astrocytes ( n = 6 biologically independent samples). c In vitro AIG assay demonstrates ectopic expression of CDKN2B or SPOCK2 leading to decreased colony numbers of LFS astrocytes. All colonies are counted and measured after 2-month culture ( n = 6 biologically independent samples). d In vivo mouse xenograft study shows ectopic expression of CDKN2B or SPOCK2 abrogating LNZ308-p53(G245D) tumor growth ( n = 3 biologically independent mice). The sizes of the tumors were measured at the indicated time. e Knockdown of CDKN2B or SPOCK2 rescues YTHDF2 depletion-induced growth inhibition in LFS astrocytes ( n = 6 biologically independent samples). f In vitro AIG assay demonstrates knockdown of CDKN2B or SPOCK2 rescuing in vitro colony formation of YTHDF2-depleted LFS astrocytes. All colonies are counted and measured after 2-month culture ( n = 6 biologically independent samples). g Xenograft study indicates that knockdown of CDKN2B or SPOCK2 rescues YTHDF2 knockdown-induced LNZ308-p53(G245D) tumor growth inhibition in nude mice ( n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. h Transcriptome analysis of CDKN2B-restored LFS astrocytes. Bubble plot for visualizing enriched GO and KEGG pathway analyses of differentially upregulated genes in CDKN2B-restored LFS astrocytes. X axis label represents the enrichment factor (number of differentially expressed genes enriched in the pathway/total number of genes in the pathway) and Y axis label represents GO annotation and KEGG pathway. Size and color of the bubble represent number of differentially expressed genes enriched in the GO or KEGG pathways and enrichment significance ( P value), respectively. The data are presented as the mean ± SEM; two-way ANOVA with Bonferroni’s multiple comparison test ( a – g ). * P < 0.05, *** P < 0.001. Source data and exact P values are provided in the Source Data file.

Article Snippet: Antibodies against p53 (1:1000, Santa Cruz, sc-126), SOX2 (1:200, Santa Cruz, sc-17320), SOX10 (1:200, Santa Cruz, sc-17342), OLIG2 (1:200, R&D Systems, BAF2418), GFAP (1:200, BioLegend, 837201), β-TUBULIN III (1:200, Sigma-Aldrich, SAB4200715), NESTIN (1:200, BioLegend, 655102), Ki67 (1:200, Life Technologies, 14-5698-82), VCL (1:2000, Sigma-Aldrich, V4505), PAX6 (1:200, Biolegend, 901301), Flag (1:2000, Sigma-Aldrich, F1804), HA (1:2000, Roche, 11666606001), V5 (1:2000, Thermo Fisher Scientific, R960-25), GFP (1:2000, Santa Cruz, sc-9996), m 6 A (1:1000, Synaptic Systems, 202003), YTHDF2 (1:1000, Proteintech, 24744-1-AP; 1:200, Aviva, ARP67917_P050), SVIL (1:500, Sigma-Aldrich, S8695), STEM121 (1:200, Takara Bio, Y40410), human nuclear antigen (1:200, Novus Biologicals, NBP2-34342), MLL1 (1:500, Bethyl Laboratories, A300-086A), H3K4me3 (1:200, Abcam, ab8580), H3K27Ac (1:200, Abcam, ab4729), METTL3 (1:1000, Proteintech, 15073-1-AP), METTL14 (1:1000, Cell Signaling, 48699), WTAP (1:1000, ABclonal Technology, A14695), FTO (1:1000, ABclonal Technology, A3851), ALKBH5 (1:1000, ABclonal Technology, A11684), CDKN2B (1:200, Thermo Fisher Scientific, MA1-12294), and SPOCK2 (1:200, Bioss Antibodies, BS-11966R) were purchased from the indicated suppliers.

Techniques: In Vivo, Knockdown, Expressing, In Vitro, Inhibition, Comparison

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a Elevated YTHDF2 expression is observed in LFS stromal cells with a heterozygous M133T mutation but not LFS stromal cells with a heterozygous 12141delG frameshift mutation compared with WT stroma ( n = 3 biologically independent samples). The data are presented as the mean ± SEM; unpaired two-tailed Student’s t test. * P < 0.05. b Box plots of TCGA RNA expression profiles (log2) in TCGA tumors with p53 WT or p53 hotspot missense mutations in LGG, GBM, BRCA, and READ specimens. Two-sided Mann Whitney Wilcoxon test is performed to compute significance. Tumors with a p53 hotspot missense mutation demonstrate decreased p21 and PUMA mRNA expression but elevated Y THDF2 mRNA expression. Box edges delineate lower and upper quartiles, the center line represents the median, and whiskers extend to 1.5 times the interquartile range. c Kaplan–Meier curves compare survival in LGG and GBM specimens with high or low levels of YTHDF2-targeted transcripts. Log-rank (Mantel–Cox) test is performed to compute significance. d Estimated hazard ratios and 95% confidence intervals for TCGA LGG and GBM patients expressing high levels of 20 YTHDF2-targeted genes. High expression of 13 of these 20 genes is positively associated with lower hazard ratios and increased survival with FDR q -value less than 5%. e A model linking the mutant p53/SVIL/MLL1 transcriptional regulatory complex to epitranscriptomic changes driving gliomagenesis. In our proposed model, mutant p53 interacts with SVIL, recruits MLL1 to the YTHDF2 promoter, and then induces YTHDF2 transcription. Elevated YTHDF2 downregulates numerous m 6 A-marked transcripts, including CDKN2B and SPOCK2 , promotes neoplastic transformation and initiates gliomagenesis. MLL1 inhibitors selectively suppress YTHDF2 expression, LFS and mutant p53 cell survival, and neoplastic transformation. Source data and exact P values are provided in the Source Data file.

Article Snippet: Antibodies against p53 (1:1000, Santa Cruz, sc-126), SOX2 (1:200, Santa Cruz, sc-17320), SOX10 (1:200, Santa Cruz, sc-17342), OLIG2 (1:200, R&D Systems, BAF2418), GFAP (1:200, BioLegend, 837201), β-TUBULIN III (1:200, Sigma-Aldrich, SAB4200715), NESTIN (1:200, BioLegend, 655102), Ki67 (1:200, Life Technologies, 14-5698-82), VCL (1:2000, Sigma-Aldrich, V4505), PAX6 (1:200, Biolegend, 901301), Flag (1:2000, Sigma-Aldrich, F1804), HA (1:2000, Roche, 11666606001), V5 (1:2000, Thermo Fisher Scientific, R960-25), GFP (1:2000, Santa Cruz, sc-9996), m 6 A (1:1000, Synaptic Systems, 202003), YTHDF2 (1:1000, Proteintech, 24744-1-AP; 1:200, Aviva, ARP67917_P050), SVIL (1:500, Sigma-Aldrich, S8695), STEM121 (1:200, Takara Bio, Y40410), human nuclear antigen (1:200, Novus Biologicals, NBP2-34342), MLL1 (1:500, Bethyl Laboratories, A300-086A), H3K4me3 (1:200, Abcam, ab8580), H3K27Ac (1:200, Abcam, ab4729), METTL3 (1:1000, Proteintech, 15073-1-AP), METTL14 (1:1000, Cell Signaling, 48699), WTAP (1:1000, ABclonal Technology, A14695), FTO (1:1000, ABclonal Technology, A3851), ALKBH5 (1:1000, ABclonal Technology, A11684), CDKN2B (1:200, Thermo Fisher Scientific, MA1-12294), and SPOCK2 (1:200, Bioss Antibodies, BS-11966R) were purchased from the indicated suppliers.

Techniques: Expressing, Mutagenesis, Two Tailed Test, RNA Expression, MANN-WHITNEY, Transformation Assay

Journal: Cancers

Article Title: Differential Expression of BOC , SPOCK2 , and GJD3 Is Associated with Brain Metastasis of ER-Negative Breast Cancers

doi: 10.3390/cancers13122982

Figure Lengend Snippet: Differentially expressed genes in primary breast cancer samples with or without brain metastasis. ( a ) Clustered heatmap of the 55 significantly expressed genes ( p < 0,01, univariate test) in fresh-frozen primary human breast cancer samples associated with the development of brain metastases (left) and breast cancer samples associated with metastasis to other organs (right); ( b ) volcano plot representation of the significantly expressed genes ( p < 0,01) clustered in ( a ); ( c ) mean normalized Log 2-transformed BOC, SPOCK2, and GJD3 differential expression between BM+ and BM− groups from the discovery sample set.

Article Snippet: The following commercially available exon-spanning TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) were used: BOC, exon 4-5 (Hs00264408_m1), GJD3/CX30.2, exon 1-1 (Hs00987388-s1), SPOCK2, exon 4-5 (Hs00360339_m1), HPRT1, exon 2-3 (Hs02800695_m1), and HMBS, exon 13-14 (Hs00609296_g1).

Techniques: Transformation Assay, Quantitative Proteomics

Journal: Cancers

Article Title: Differential Expression of BOC , SPOCK2 , and GJD3 Is Associated with Brain Metastasis of ER-Negative Breast Cancers

doi: 10.3390/cancers13122982

Figure Lengend Snippet: Relative mRNA expression of BOC and SPOCK2 in primary breast cancers of patients who developed metastasis to organs excluding brain (BM−) and including brain (BM+). Bars indicate Mean ± SD.

Article Snippet: The following commercially available exon-spanning TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) were used: BOC, exon 4-5 (Hs00264408_m1), GJD3/CX30.2, exon 1-1 (Hs00987388-s1), SPOCK2, exon 4-5 (Hs00360339_m1), HPRT1, exon 2-3 (Hs02800695_m1), and HMBS, exon 13-14 (Hs00609296_g1).

Techniques: Expressing

Journal: Cancers

Article Title: Differential Expression of BOC , SPOCK2 , and GJD3 Is Associated with Brain Metastasis of ER-Negative Breast Cancers

doi: 10.3390/cancers13122982

Figure Lengend Snippet: Box-whisker plots of ( a ) BOC -, ( b ) SPOCK2 -, and ( c ) GJD3 -IHC mean intensities, with tabular statistics summary for both BM+ and BM− groups of ER- primary breast cancers. The lowest and highest boundaries of the box represent the 25th and 75th percentiles, respectively. The solid line across the box indicates the median value. Error bars indicate the 5th–95th percentile.

Article Snippet: The following commercially available exon-spanning TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) were used: BOC, exon 4-5 (Hs00264408_m1), GJD3/CX30.2, exon 1-1 (Hs00987388-s1), SPOCK2, exon 4-5 (Hs00360339_m1), HPRT1, exon 2-3 (Hs02800695_m1), and HMBS, exon 13-14 (Hs00609296_g1).

Techniques: Whisker Assay

Journal: Cancers

Article Title: Differential Expression of BOC , SPOCK2 , and GJD3 Is Associated with Brain Metastasis of ER-Negative Breast Cancers

doi: 10.3390/cancers13122982

Figure Lengend Snippet: Representative BOC , SPOCK2 , and GJD3 positive and negative stainings of FFPE primary breast cancer samples. On the left column ( a , c , e ) are representative positive stainings from BM+ group; on the right column ( b , d , f ) are representative negative stainings from BM− group. Scale bars = 50 µm.

Article Snippet: The following commercially available exon-spanning TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) were used: BOC, exon 4-5 (Hs00264408_m1), GJD3/CX30.2, exon 1-1 (Hs00987388-s1), SPOCK2, exon 4-5 (Hs00360339_m1), HPRT1, exon 2-3 (Hs02800695_m1), and HMBS, exon 13-14 (Hs00609296_g1).

Techniques:

Journal: Scientific Reports

Article Title: MicroRNA-127 targeting of mitoNEET inhibits neurite outgrowth, induces cell apoptosis and contributes to physiological dysfunction after spinal cord transection

doi: 10.1038/srep35205

Figure Lengend Snippet: ( a ) Venn diagram of target genes of miR-127 which derived from conventional online programs of miRDB, TargetScan, miRNAWalk and miRGen. MitoNEET, KCC1 and Spock2 are the common targets of miR-127 in the four conventional online programs which were selected for further investigation. ( b ) The alignment of the seed regions of miR-127 with MitoNEET, KCC1 and Spock2 3′-UTR. ( c ) Alignment of the seed regions of miR-127 with MitoNEET and KCC1 3′ UTR, and the mutation of MitoNEET and KCC1 3′ UTR sequence in the complementary site. ( d,e,f ) 293Tα cells were co-transfected with 80 nM of miR-127 mimic or NS-miRNA and 100 ng/ml of 3′-UTR reporter plasmid of MitoNEET, KCC1 and Spock2 or the relative mutant form. Luciferase activity was detected at 48 h after transfection. Relative luciferase activity was calculated with (Rluc miRNA/hLuc miRNA)/(Rluc NS-miRNA/hLuc NS-miRNA). ( g,h ) Total RNA of primary spinal neurons was extracted and performed for qRT-PCR of MitoNEET ( g ) and KCC1 ( h ) 72 h after being transfected with miR-127 mimic or NS-miRNA. ( i,j ) The protein expression of MitoNEET ( i ) and KCC1 ( j ) in primary spinal neurons was detected by ELISA 72 h after being transfected with miR-127 mimic or NS-miRNA. *P < 0.05, **P < 0.01 compared with NS-miRNA.

Article Snippet: Then, 293Tα cells (5 × 10 5 cells per well) were seeded into triplicate wells of 6-well plates and allowed to settle for 12 h. The 3′ UTR luciferase plasmids of MitoNEET, KCC1 and Spock2 or control reporter plasmid (100 ng/ml, Guangzhou RiboBio, China) were transfected into 293Tα cells using SuperFectinTM II In Vitro DNA Transfection Reagent (Pufei Biotech, China).

Techniques: Derivative Assay, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: MicroRNA-127 targeting of mitoNEET inhibits neurite outgrowth, induces cell apoptosis and contributes to physiological dysfunction after spinal cord transection

doi: 10.1038/srep35205

Figure Lengend Snippet: ( a,b ) Double-label fluorescence detection of NeuN/MitoNEET ( a ) and NeuN/KCC1 ( b ) were carried out in spinal cord. Sections were stained with DAPI ( a , b , blue, the first panel) to show all nuclei, NenN ( a , b , green, the second panel), MitoNEET ( a , red, the third panel), KCC1 ( b , red, the third panel), and the merge image ( a,b , the last panel). The merge image shows the region of co-localization appearing yellow. ( c,d ) Double-label fluorescence detection of GFAP/MitoNEET ( c ) and GFAP/KCC1 ( d ) were carried out in spinal cord. Sections were stained with DAPI ( c , d , blue, the first panel) to show all nuclei, GFAP ( c , d , green, the second panel), MitoNEET ( c , red, the third panel), KCC1 ( d , red, the third panel), and the merge image ( c , d , the last panel). The merge image shows the region of co-localization appearing yellow. Scale bar: ( a–d ) 50 μm.

Article Snippet: Then, 293Tα cells (5 × 10 5 cells per well) were seeded into triplicate wells of 6-well plates and allowed to settle for 12 h. The 3′ UTR luciferase plasmids of MitoNEET, KCC1 and Spock2 or control reporter plasmid (100 ng/ml, Guangzhou RiboBio, China) were transfected into 293Tα cells using SuperFectinTM II In Vitro DNA Transfection Reagent (Pufei Biotech, China).

Techniques: Fluorescence, Staining

Journal: Scientific Reports

Article Title: Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion

doi: 10.1038/s41598-018-27180-z

Figure Lengend Snippet: FunRich analysis of iTRAQ data. ( A ) Proteins with a ≥ 2-fold increase in expression and ( B ) a ≥ 2-fold decrease in expression when compared to RWPE1 EVs. Venn diagrams were created using the Public Research Centre for Health’s Venn diagram generator tool ( http://www.bioinformatics.lu/venn.php ). ( C ) Western blot analysis of Actin, Filamin-A, TGFBI, HSPA5 and SPOCK2 confirmed iTRAQ quantitated values. ( C ) Enriched biological pathways for all detected EV proteins. Degradation pathways were the predominant feature in the total dataset. ( D ) Enriched biological pathways for CD9 low EV proteins with a ≥ 2-fold increase in expression to RWPE1 EVs. ( E ) Enriched biological pathways for CD151 high EV proteins with a ≥ 2-fold increase in expression to RWPE1 EVs. ( F ) Enriched biological pathways for WPE1-NB26 EV proteins with a ≥ 2-fold increase in expression to RWPE1 EVs. Graphs were created using FunRich v2.1.2. which converts protein IDs to gene names. Western blot images are cropped, with full-length blots presented in Supplementary Fig. .

Article Snippet: Commercial antibodies were purchased from the following: Rabbit-anti-GAPDH (BioVision #3777R-100; Sapphire Biosciences, Redfern, NSW, Australia), mouse-anti-CD63 (BioVision #A1502-50), mouse-anti-CD82 [TS82b] (Abcam #ab59509; Abcam Australia, Melbourne, VIC, Australia), rabbit polyclonal HSPA5 (GeneTex #GTX127934; Sapphire Biosciences), FLNA (GeneTex #GTX109931), SPOCK2 (GeneTex #GTX121068) and TGFBI (GeneTex #GTX100744).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion

doi: 10.1038/s41598-018-27180-z

Figure Lengend Snippet: Comparison of iTRAQ and western blot quantitated values.

Article Snippet: Commercial antibodies were purchased from the following: Rabbit-anti-GAPDH (BioVision #3777R-100; Sapphire Biosciences, Redfern, NSW, Australia), mouse-anti-CD63 (BioVision #A1502-50), mouse-anti-CD82 [TS82b] (Abcam #ab59509; Abcam Australia, Melbourne, VIC, Australia), rabbit polyclonal HSPA5 (GeneTex #GTX127934; Sapphire Biosciences), FLNA (GeneTex #GTX109931), SPOCK2 (GeneTex #GTX121068) and TGFBI (GeneTex #GTX100744).

Techniques: Western Blot

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: Induction of SPOCK2 expression by IFNs, poly(I·C), and virus. (A and B) Nine-week-old mice were infected with 100 or 500 PFU of PR8 IAV. (A) The expression of SPOCK2 mRNA in the lungs of infected mice was detected by quantitative RT-PCR (qRT-PCR). The experiment was performed in triplicate, and the mean fold is indicated relative to the control group. Each bar indicates the average value ± SD obtained from triplicate experiments. Statistically significant differences from the controls, calculated using the t test, are indicated as follows: *, P < 0.05, and **, P < 0.01. (B) Proteins in the lungs of infected mice were detected by immunoblotting with anti-SPOCK2 and anti-GAPDH. Intensity values were quantified using the Multigauge program (Fuji Film). Each bar indicates the average value ± SD obtained from triplicate experiment. Statistically significant differences from the controls, calculated using the t test, are indicated as follows: *, P < 0.05, and ***, P < 0.001. P.I., days postinfection. (C) A549 cells were infected with PR8 IAV at the indicated dose (top) or for the indicated time (bottom). Immunoblots of cell extracts were probed with anti-SPOCK2 and with anti-GAPDH antibodies as a loading control. (D and E) A549 cells were transfected with 1 μg of poly(I·C) (D) or incubated with IL-1β (20 ng/ml), IL-6 (20 ng/ml), TNF-α (20 ng/ml), or TGF-β (20 ng/ml) (E) at the indicated time. Cell extracts were immunoblotted with anti-SPOCK2 and anti-GAPDH antibodies used as a loading control. (F, K, and L) Indicated subtypes of SPOCK mRNA levels were measured by qRT-PCR at the indicated time points after treatment with human IFN-α (100 U/ml) or IFN-β (100 U/ml) in A549 cells. Expression was normalized to that of GAPDH. Each bar indicates the average value ± SD obtained from triplicate experiments. *, P < 0.05 compared with the control (t test). (G) A594 cells were treated with human IFN-α (100 U/ml) or IFN-β (100 U/ml) for the indicated time. Cell extracts were immunoblotted with anti-SPOCK2 and with anti-GAPDH antibodies as a loading control. (H) A549 cells were transfected with control siRNA or IFN-αR1 siRNA. After 20 h, the cells were infected with PR8 IAV (MOI, 1) for 12 h. SPOCK2, IFN-αR1, and GAPDH levels were determined by qRT-PCR. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for the control and SPOCK2 siRNA-transfected cells (t test). (I and J) (Left) Diagram of the pSPOCK2-luc construct, the 0.5-kb genomic DNA fragment of SPOCK2, with or without predicted STAT binding sites. (Right) A549 cells were transfected with pSPOCK2-luc and pRL-TK. After 36 h of transfection, the A549 cells were treated with human IFN-α (100 U/ml) or IFN-β (100 U/ml). After 12 h, the dual-luciferase assay was performed. “Mock” indicates samples treated with distilled water-treated. The graphs present the average values from triplicate experiments. Error bars represent SDs. *, P < 0.05 compared to mock (t test).

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Construct, Binding Assay, Luciferase

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: SPOCK2 inhibits influenza virus infection. (A) A549 cells were transfected with V5-tagged SPOCK2 or empty vector. After 24 h, the cells were infected with PR8 IAV (MOIs, 0.01, 0.1, and 1) for the indicated times. HA vRNA levels were measured by qRT-PCR. Expression was normalized to GAPDH. In the graph, HA expression at 6 h in the EMPTY-V5-transfected sample was set to 1 at each dose. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparison between EMPTY-V5- and SPOCK2-V5-transfected cells (t test). (B) A549 cells were transfected with control siRNA or SPOCK2 siRNA. After 24 h, the cells were infected with PR8 IAV (MOI, 0.01, 0.1, and 1) for the indicated times. After another 24 h, HA vRNA and GAPDH levels were determined by qRT-PCR. In the graph, HA expression at 6 h in the control siRNA-transfected sample was set to 1 at each dose. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P < 0.05; **, P < 0.01 for comparison between the control and SPOCK2 siRNA-transfected cells (t test). (C and D) Cell lysates from SPOCK2-V5-overexpressing (C) or SPOCK2-silenced (D) cells infected with PR8 IAV for 12 h (MOI, 1) were analyzed with anti-NP and anti-V5, anti-SPOCK2, and anti-GAPDH as a loading control. (E and F) At 36 h posttransfection, A549 cells were infected with IAV at an MOI of 1 for 24 h or 48 h. The supernatants from infected A549 cells were collected, and the viral titers were measured by the TCID50 method. A549 cells were transfected with the V5-tagged SPOCK2 plasmid and the indicated siRNA (F). The bars indicate the mean values ± SDs obtained from three experiments. (G and H) A549 cells (G) and HEK293 cells (H) were transfected with control siRNA or SPOCK2 siRNA. After 24 h, the cells were infected for 12 h after treatment with IFN-α (100 U) for 16 h. HA vRNA levels were measured by qRT-PCR. Expression was normalized to that of GAPDH. Each graph presents mean data from three independent experiments, and error bars indicate SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P > 0.05 (not significant) (t test).

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Infection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: Recombinant SPOCK-2 protein inhibits virus infection in vitro and in vivo. (A) Schema of the experimental procedure. (B to D) A549 cells were infected with PR8 IAV (MOI, 0.1) in the presence of medium from SPOCK2-overexpressing cells. After 12 h, the cells under the indicated conditions were collected and HA vRNA expression was detected by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. *, P < 0.05; **, P < 0.01. (E) Purified SPOCK2 from lysate or culture medium was immunoblotted with anti-His. (F) A549 cells were infected with PR8 IAV (MOI, 0.1) in the presence of purified SPOCK2 (20 μg) from the lysate or medium of His-tagged SPOCK-overexpressing A549 cells. After 12 h, the cells under the indicated conditions were collected, and HA vRNA expression was detected by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. *, P < 0.05; **, P < 0.01 (t test). (G to J) Mice were intranasally infected with PR8 IAV at 50 PFU and treated with buffer or purified SPOCK2 at 10 μg per mouse. (G) Body weight changes in PR8 IAV virus-infected mice treated with buffer or purified SPOCK2 were monitored. The graph presents the average values ± SDs (n = 6 for each group). (H) Representative hematoxylin and eosin (H&E) histology of lung tissues from the three mice in panel G evaluated at 7 days postinfection (dpi). The enlarged image shows the bronchiole region. Arrows indicate infiltration of inflammatory cells. (I) The intracellular copy numbers of the PR8 IAV HA gene and IFN-β in lung tissues at 7 dpi from the mice in panel G were measured by qRT-PCR. Each bar indicates the average value ± SD (n = 3). (J) The intracellular amount of the PR8 IAV NP gene in lung tissues at 7 dpi from the mice in panel G were measured by immunoblotting (n = 3). P values were calculated by two‐way ANOVA. **, P < 0.01; ***, P < 0.001.

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Recombinant, Infection, In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Purification, Western Blot

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: The heparan sulfate moiety is responsible for the antiviral effect of SPOCK2. (A) Domain deletion mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. (B) Site-directed mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. For the bottom portion, cell lysates from the top portion were analyzed with anti-M1 and anti-V5, and anti-GAPDH was detected as a loading control. (C) At 36 h posttransfection, A549 cells were infected with IAV at an MOI of 1 per well for 24 h or 48 h. The supernatants from cells infected with A549 cells were collected, and the viral titers were measured by the TCID50 method. The bars indicate the mean values ± SDs obtained from three experiments. (D) A549 cells were transfected with EXT1, -2, and -3 siRNAs and a V5-tagged SPOCK2-expressing construct or empty vector. HA vRNA levels were measured by qRT-PCR. The expression was normalized by GAPDH (upper graph). The silencing efficiencies of EXT1, EXT2, and EXTL3 were measured by qRT-PCR (lower graph). Expression was normalized by GAPDH. All graphs indicate the average values ± SDs obtained from triplicate experiments. ***, P < 0.001 (t test).

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Infection, Quantitative RT-PCR, Transfection, Expressing, Construct, Plasmid Preparation

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: Purified SPOCK2 inhibits attachment and entry of influenza virus. (A) A549 cells were infected with DiI-labeled PR8 IAV with buffer or 20 μg of purified SPOCK2 for 20 min. DiI and nucleus (Hoechst) staining were examined by confocal microscopy. Representative images are shown. Scale bars, 200 μm. DIC, differential interference contrast. (B) A549 cells were infected with PR8 IAV in the presence of purified SPOCK2 (20 μg) for 20 min and then processed for subcellular fractionation. The fractions from different compartments were used for RNA isolation and the determination of HA vRNA levels by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. P values were calculated by two‐way ANOVA. *, P < 0.05. (C) An HI assay was performed using turkey RBCs. The RBCs were added to a 96-well v-bottom plate. PR8 IAV was added with serially diluted purified SPOCK2 and incubated for 1 h at room temperature. The inhibition of PR8 IAV attachment to RBCs was then measured by observing each well. (D) Plaque assays of PR8 IAV with buffer or purified SPOCK2 (20 μg). (E) HEK293T cells were transfected with PB1, PB2, PA, and eGFP plasmid carrying NS1 vRNA with or without NP plasmid. After 48 h, the relative mean fluorescence intensity of GFP was measured by FACS. All graphs indicate the average values ± SDs obtained from triplicate experiments. ***, P < 0.001. (F) A549 cells were transfected with NP, PB1, PB2, PA, an eGFP plasmid carrying NS1 vRNA and a V5-tagged SPOCK2 expression construct or empty vector. After 48 h, the mean fluorescence intensity of GFP was measured by FACS. NT, transfection of reporter segment alone. Each bar indicates the average value ± SD obtained from triplicate experiments. #, P > 0.05 (not significant).

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Purification, Infection, Labeling, Staining, Confocal Microscopy, Fractionation, Isolation, Quantitative RT-PCR, HI Assay, Incubation, Inhibition, Transfection, Plasmid Preparation, Fluorescence, Expressing, Construct

Journal: Journal of Virology

Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

doi: 10.1128/JVI.00662-19

Figure Lengend Snippet: Influenza virus preferentially binds the sialic acid moiety of SPOCK2. (A and B) A549 cells treated with tunicamycin for 12 h (A) or PNGase F (250 U) (B) were subjected to immunoblot analysis with anti-SPOCK2, and anti-GAPDH was detected as a loading control. (C) Lysates of empty vector- or NA-expressing A549 cells infected with PR8 IAV for 12 h were subjected to immunoblot analysis with anti-SPOCK2 and anti-NA. Anti-GAPDH was detected as a loading control. (D) Purified SPOCK2 (1 μg) was incubated with PBS or PR8 IAV for 1 h and subjected to immunoblot analysis with anti-His and anti-M1. (E and F) Purified SPOCK2 (1 μg) from SPOCK2-overexpressing cells (E) or NA-co-overexpressing cells (F) analyzed by Western blotting and SNA blotting. (G) An HI assay was performed using turkey RBCs. The RBCs were added to a 96-well v-bottom plate. PR8 IAV was added with serially diluted purified SPOCK2 from the medium of SPOCK2-overexpressing cells or NA-co-overexpressing cells and incubated for 1 h at room temperature. The inhibition of PR8 IAV attachment to RBCs was then measured by observing each well. (H) Schematic illustration of the proposed model.

Article Snippet: This 6× His-containing human SPOCK2 expression plasmid was transfected into mammalian cells (HEK293T or CHO DG44) with polyethylenimine (PEI; Polysciences).

Techniques: Western Blot, Plasmid Preparation, Expressing, Infection, Purification, Incubation, HI Assay, Inhibition