Journal: bioRxiv

Article Title: Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signalling pathway

doi: 10.1101/2022.11.28.518246

Figure Lengend Snippet: a-b Levels of endogenous iRhom2 were analysed by immunoblotting in lysates from cells treated with 200 nM PMA for the indicated time intervals ( a ) or after transfection for 72 h with either control siRNA or siRNA against SEC11A and SEC11C ( b ). c-d Fold change of gene expression from a published RNA-Seq dataset of iRhom1 (RHBDF1), iRhom2 (RHBDF2) and all subunits of SPC (SEC11A, SEC11B, SPCS1, SPCS2, SPCS3) in lesional psoriasis compared to control ( c ) or lesional psoriasis compared non-lesional psoriasis ( d ). e-f Venn diagrams showing overlap between differentially expressed genes found in a published RNA-Seq dataset of lesional psoriasis and upregulated ( e ) or downregulated ( f ) at 3 h induction of iR2-1-374 (top). Bar graphs showing summary of the GO enrichment analysis on the set of common upregulated (n=104) or downregulated (n=149) genes between lesional psoriasis and iR2-1-374. FDR: False discovery rate. Different colours denote range of fold enrichment (FE).

Article Snippet: For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, #sc-47778; 1:5000), FLAG-HRP (Sigma Aldrich, #A8592; 1:4000), HA-HRP (Roche, #11867423001; 1:2000), KDEL (AbCam, #ab12223; 1:2000), iRhom2-NT-specific (( ); 1:500), Myc (Abcam, #ab9132; 1:2000), SEC11A (Proteintech, #14753-1-AP; 1:500), SEC11C (Novus Biologicals, NBP1-80774; 1:500), SPCS1 (Proteintech, #11847-1-AP; 1:500), SPCS2 (Merck Life Science, #HPA013386; 1:500), SPCS3 (Santa Cruz, sc-377334; 1:500), RHBDL4 (( ); 1:1000), POL2 (MBL International, MABI0601; 1:1000), TFIID (Santa Cruz, #sc-273; 1:1000), Histone H3 (Cell Signaling, #4499T; 1:4000), GRIN2D (Novus Biologicals, NBP2-94573; 1:1000), ZNF195 (Novus Biologicals, NBP2-93054; 1:1000), DDR1 (Cell Signaling, #5583T ; 1:1000), DDX3X (Cell Signaling, #3189S; 1:1000), ADAM17 (AbCam, #ab39162; 1:2000).

Techniques: Western Blot, Transfection, Control, Gene Expression, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: Multiple alignments of Arxes1, Arxes2 and Spcs3 promoters and transcripts

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: mRNA expression patterns of Arxes1, Arxes2 and Spcs3 during in vitro adipogenesis, in mouse tissues and upon in vitro and in vivo rosiglitazone treatment. ( A–C ) Shown are qPCR measurements during the course of adipogenesis in three different cell models. Cells were induced with DMI cocktails 2 days post confluence (d0) as specified in ‘Materials and Methods’ section. Values are expressed relative to d0 measurements. Data are presented as mean ± SEM from two independent MEF isolations or two and three experiments for OP9 and 3T3-L1 cells, respectively. ( D ) Distribution of mRNA expression in metabolically relevant tissues in wild-type mice. Expression is shown relative to heart as fold-expression. Inset represents a scale-up of the Spcs3 expression values. Data are presented as mean ± SEM ( n = 3). Abbreviations: WAT, white adipose tissue (epididymal); BAT, brown adipose tissue (interscapular); L, liver; SM, skeletal muscle; H. heart. ( E ) 3T3-L1 adipocytes were treated with 1 µM rosiglitazone at Day 7 of differentiation. RNA was harvested 24 h after rosiglitazone treatment or medium change (DMEM) and measured with qPCR using the indicated primers. Data are represented as mean ± SEM from three independent experiments. Student’s t -test: ** P < 0.01. ( F ) Male mice were kept on a rosiglitazone-containing chow diet (0.01% w/w) or on normal chow for 7 weeks post-weaning. Prior to harvesting epididymal fat pads mice were fasted for ∼4 h to synchronize nutritional states. Data are represented as mean ± SEM ( n = 5–6). Student’s t -test: ** P < 0.01.

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Expressing, In Vitro, In Vivo, Metabolic Labelling

Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: Silencing of the Arxes abrogates adipogenesis, while Spcs3 knockdown does not change adipogenic marker gene expression. 3T3-L1 cells were transduced with silencing constructs (siArxes or siSpcs3) or nontargeting control (ntc), and induced to undergo adipogenesis with standard DMI treatment unless indicated otherwise. ( A ) qPCR mRNA measurement shows that Arxes knockdown using the siArxes silencing constructs is specific to the Arxes, while Spcs3 expression is not reduced. Data are presented as mean ± SEM from three independent transduction experiments. ( B ) Lipid droplets were visualized by oil red O staining of 7 days differentiated 3T3-L1 cells. Lower panel of the oil red O staining shows a partial rescue of the differentiation deficiency if 1 µM rosiglitazone is present during differentiation procedure. ( C ) Expression of marker genes of adipogenesis was measured with qPCR on Day 7 of adipogenesis in Arxes-silenced and control cells. Data are presented as mean ± SEM from three independent transduction experiments. ( D ) qPCR mRNA measurement shows that Spcs3 knockdown using the siSpcs3 silencing construct is specific to the Spcs3 while Arxes mRNA is not reduced. Data are presented as mean ± SEM from two independent transduction experiments. ( E ) Spcs3 knockdown does not influence adipogenic marker gene expression as measured with qPCR on Day 7 in DMI induced cells. Data are presented as mean ± SEM from two independent transduction experiments.

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Knockdown, Marker, Gene Expression, Transduction, Construct, Control, Expressing, Staining