Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) The genomic structure of the NMNAT2/SMG7 region and positions of genetic variants are indicated. (B) The allelic P value (−log10P value) of each genetic variant with SLE is plotted against its position as a circle (genotyped) or a triangle (imputed) for European American (EA), Amerindian/Hispanic (HS), African American (AA) and Asian (AS), respectively. Genetic variants are highlighted using different colors according to their strength of linkage disequilibrium (r2) with rs2022013 (shown as a blue diamond) in each ancestry. The dashed line represents a Bonferroni corrected P<1×10−3. Arrows identify rs2022013 and SNPs demonstrating the most significant association signals in each ancestry. Black rectangle identifies group 3 SNPs at SMG7 strongly associated with SLE in EA and the best-associated SNP rs2702178 is indicated. (C) Trans-ancestral meta-analysis is conducted on 8 SNPs that remain significant associations after Bonferroni correction in both EA and HS. Black rectangle identifies group 1&2 SNPs at NMNAT2 intron 1 showing Pmeta values exceeding the GWAS significance level. The dashed line represents the significance level of 5×10−8.

Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Variant Assay

Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) Association of rs2275675 genotypes with SMG7 mRNA levels in PBMCs from European-derived SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B&C) Allelic differences of rs2275675 and rs10911339 in luciferase activity. Data show the mean ± SEM results of three independent experiments.

Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Derivative Assay, Luciferase, Activity Assay

Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) SMG7 mRNA levels in PBMCs correlated inversely with ANA titers of SLE patients. Among 68 patients, 56 were recruited at UCLA and 12 were recruited at OMRF, and their ANA titers were measured in Clinical Laboratory at each site. (B&C) Increased ANA IgG and CCL19 levels after SMG7 silencing in SLE PBMCs. PBMCs from ANA positive SLE patients (n=13) were incubated for 5 days in the presence or absence of siRNA targeting SMG7, GAPDH or siRNA with a non-targeting sequence (NC), respectively. ANA IgG (B) and CCL19 (C) levels in culture supernatants were measured by ELISA, and plotted as fold change with respect to mock (culture medium only). Results are presented as mean ± SEM.

Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Incubation, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) The genomic structure of the NMNAT2/SMG7 region and positions of genetic variants are indicated. (B) The allelic P value (−log10P value) of each genetic variant with SLE is plotted against its position as a circle (genotyped) or a triangle (imputed) for European American (EA), Amerindian/Hispanic (HS), African American (AA) and Asian (AS), respectively. Genetic variants are highlighted using different colors according to their strength of linkage disequilibrium (r2) with rs2022013 (shown as a blue diamond) in each ancestry. The dashed line represents a Bonferroni corrected P<1×10−3. Arrows identify rs2022013 and SNPs demonstrating the most significant association signals in each ancestry. Black rectangle identifies group 3 SNPs at SMG7 strongly associated with SLE in EA and the best-associated SNP rs2702178 is indicated. (C) Trans-ancestral meta-analysis is conducted on 8 SNPs that remain significant associations after Bonferroni correction in both EA and HS. Black rectangle identifies group 1&2 SNPs at NMNAT2 intron 1 showing Pmeta values exceeding the GWAS significance level. The dashed line represents the significance level of 5×10−8.

Article Snippet: Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Variant Assay

Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) Association of rs2275675 genotypes with SMG7 mRNA levels in PBMCs from European-derived SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B&C) Allelic differences of rs2275675 and rs10911339 in luciferase activity. Data show the mean ± SEM results of three independent experiments.

Article Snippet: Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Derivative Assay, Luciferase, Activity Assay

Journal: Annals of the rheumatic diseases

Article Title: Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

doi: 10.1136/annrheumdis-2015-208441

Figure Lengend Snippet: (A) SMG7 mRNA levels in PBMCs correlated inversely with ANA titers of SLE patients. Among 68 patients, 56 were recruited at UCLA and 12 were recruited at OMRF, and their ANA titers were measured in Clinical Laboratory at each site. (B&C) Increased ANA IgG and CCL19 levels after SMG7 silencing in SLE PBMCs. PBMCs from ANA positive SLE patients (n=13) were incubated for 5 days in the presence or absence of siRNA targeting SMG7, GAPDH or siRNA with a non-targeting sequence (NC), respectively. ANA IgG (B) and CCL19 (C) levels in culture supernatants were measured by ELISA, and plotted as fold change with respect to mock (culture medium only). Results are presented as mean ± SEM.

Article Snippet: Total RNAs purified from PBMCs were reverse-transcribed into cDNA to measure levels of SMG7 , SMG7-AS1 and housekeeping gene RPLP0 by TaqMan assays ( SMG7 : Hs00539224_m1, SMG7-AS1 : Hs01069005_m1, RPLP0 : Hs99999902_m1; Life Technologies).

Techniques: Incubation, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: BMC Genomics

Article Title: ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis

doi: 10.1186/s12864-017-4285-2

Figure Lengend Snippet: Challenge of mutant cell lines to different cell death stimuli. The control (parental) cell line as well as individually engineered null mutant cell lines ( Smg7, Ces2a, Hnrnpf, Tnfrsf1a ) were compared for resistance to a panel of cell death inducing agents. Two-way, repeated-measures ANOVA with a Bonferroni post test showed Smg7 and Ces2a mutant cell lines to be statistically different from control cells at different doses (colored asterisks: above, more resistant; below, more sensitive. Statistics of other cell lines not shown). * P < .05. ** P < .01. *** P < .001. Z-Val-Ala-Asp-fluoromethylketone (zVAD) was added to control cells to demonstrate caspase-dependent cell death. Abbreviations: Deoxycholic acid (DCA); 5-Fluorouracil, (5-FU)

Article Snippet: Selected antibodies were purchased for p65 (Santa Cruz), Caspase-8 (Cell Signaling), Smg7 (Biomol), Ces2a (LSBio), PAN-actin (Cell Signaling), and p53 (Cell Signaling).

Techniques: Mutagenesis, Control

Journal: BMC Genomics

Article Title: ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis

doi: 10.1186/s12864-017-4285-2

Figure Lengend Snippet: Characterization of TNFa-induced apoptosis in Smg7 −/− , Ces2a −/− and control cell lines. a The NF-κB responsive p65 protein was detected by high-content microscopy and the ratio of nuclear:cytosolic forms was compared for the mutant cell lines in untreated and TNFa treated conditions. b The NF-κB transcriptional response was measured by luciferase assay and fold induction calculated relative to a transfection control. c Smg7 −/− , Ces2a −/−, Hnrnpf −/− cell lines can be resensitized to TNFa by addition of cycloheximide (CHX) but are significantly less sensitive than the control. Trnfrsf1a −/− cells cannot be resensitized. d Cell death proteins were detected by Western blot at 0 and 16 h of TNFa treatment. Basal levels of p53 are upregulated in Ces2a −/− and in Smg7 −/− cells following TNFa addition. e, f Control cells show a significant increase in nuclear (DAPI-stained) fraction of p53 (red) following TNFa treatment while Smg7 −/− cells show similar levels. * P < .05. ** P < .01. *** P < .001. n.s. not significant. Scale bar is 50 μm

Article Snippet: Selected antibodies were purchased for p65 (Santa Cruz), Caspase-8 (Cell Signaling), Smg7 (Biomol), Ces2a (LSBio), PAN-actin (Cell Signaling), and p53 (Cell Signaling).

Techniques: Control, Microscopy, Mutagenesis, Luciferase, Transfection, Western Blot, Staining