Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Activity Assay, Expressing, Functional Assay, Gene Expression, Quantitative RT-PCR, esiRNA

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Negative Control, Quantitative RT-PCR, Knockdown, IF-P, ChIP-qPCR, Control, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 1 Representative images of hematoxylin and eosin staining (HE) and immunohistochemistry against CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules; HEVs), CD40 (follicular dendritic cells), and BCL6 (germinal center) in a small-sized aggregation of lymphocytes in goat mammary gland tissues. Scale bar, 100 mm or 25 mm (clippings).

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 2 Representative images of hematoxylin and eosin staining (HE) staining and immunohistochemistry against CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules; HEVs), CD40 (follicular dendritic cells), and BCL6 (germinal center) in a large-sized aggregation of lymphocytes in goat mammary glands. Images of the same position are shown. Scale bar, 100 mm.

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 3 Representative images of immunohistochemistry against CD20 (B cells) and CD3 (T cells) in large aggregations of lymphocytes in goat mammary glands. Images of the same position are shown. Scale bar, 100 mm.

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Immunohistochemistry

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Verification of loss of CerS2 in CerS2 ΔIns1E cells by immunoblot. Left: representative immunoblot. Right: quantification of Cers2 signals ( n = 8 independent experiments). b , Representative immunostaining (left) and quantification (right) of ER marker PDI in control and CerS2 ΔIns1E cells ( n = 36 control versus 28 CerS2 ΔIns1E well sites from one experiment). Scale bar, 10 μm. c , Quantification of insulin content in control and CerS2 ΔIns1E cells at low (2 mM) and high (25 mM) glucose levels ( n = 5 independent experiments). d , Experimental design and results for proteome analyses in control and CerS2 ΔIns1E cells. e , Volcano plot showing log 2 fold change of proteins between CerS2 ΔIns1E and control cells plotted against the −log 10 P value of a two-sided paired Student’s t -test. BH-FDR <0.05 and fold change >1.5 was used as significance cut-offs ( n = 3 control versus 3 CerS2 ΔIns1E samples collected in three independent experiments). f , g , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of control and CerS2 ΔBKO mice. f , Representative immunoblot. Each lane represents islets of one individual mouse. g , Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six CerS2 ΔBKO mice). h , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of 12-week-old control and db/db.BKS mice. Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six db/db.BKS mice). Representative immunoblot is shown in Extended Data Fig. . i , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of 12-week-old control and ob/ob.B6 mice. Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six ob/ob.B6 mice). Representative immunoblot is shown in Extended Data Fig. . Statistical analysis was performed using a two-sided Student’s t -test ( a , b , g , h and i ) and two-way ANOVA with Sidak’s multiple comparisons test ( c ). P values are stated in each figure. Bar graphs represent mean ( c ) or mean ± s.e.m. ( a , b , g , h and i ). Connecting lines indicate both samples are from one experiment. Data points in b represent individual well sites. Data points in a and c represent independent experiments. Data points in g – i represent islets from individual mice. Stain-free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Western Blot, Immunostaining, Marker, Control, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Quantification of Pcsk1 levels in control and CerS2 ΔIns1E cells by CrispR-verified antibody (Supplementary Fig. , Cell Signaling #11914, discontinued). Left, representative immunoblot. Right, quantification of Pcsk1 signals ( n = 5 independent experiments). b , Quantification of Pcsk1 protein levels in control and CerS2 BKO islets by CrispR-verified antibody (Supplementary Fig. , Cell Signaling #11914, discontinued). Left, representative immunoblot. Right, quantification of Pcsk1 signals ( n = 8 independent experiments). c , Quantification of Pcsk1 mRNA levels in islets from male control and CerS2 BKO mice by qPCR ( n = 5 independent experiments). d , Representative immunoblots to Fig. . e , Representative immunoblots to Fig. . f, g , Quantification of mRNA levels of Pcsk1 and various CerS in islets of 12 week old control and db/db.BKS mice (f, n = 4 control and 4 db.db/BKS islet samples) and islets of 12 week old control and ob/ob.B6 mice (g, n = 4 control and 6 ob/ob.B6 islets samples). Statistical analysis was performed using a paired two-sided Student’s t -test (a, b), two-sided Student’s t -test (c) and two-sided multiple t -tests with Holm-Sidak correction (f, g). P -values are stated in each figure. Bar graphs represent means (a, b) or means + s.e.m. (c, f, g). Connecting lines indicate both samples are from one experiment. Data points represent independent experiments (a), islets from individual mice (c) or individual islet samples (f, g). Stain-Free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Control, CRISPR, Western Blot, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Immunoblot detection of human CerS6 (hCerS6) and Tmed2 protein levels in INS1E cells after overexpression of hCerS6 via adenovirus for 48 h (representative immunoblot). b , Ins1E cell counts after 48 hours of infection with a control adenovirus or hCerS6-expressing adenovirus ( n = 3 independent experiments). A reduction of cell counts is in line with the ability of CerS6 overexpression to induce apoptosis in several cell types. c , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels (Cell Signaling #18030) in Ins1E cells infected with a control adenovirus or hCerS6-expressing adenovirus for 48 h (representative immunoblot). d-g , Quantification of Tmed2 (d), Pcsk1 (e), Pro-Pcsk1 signals (f) and Pcsk1/ Pro-Pcsk1 ratio (g) from 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (b, d-g). Data points in (b, d-g) represent independent experiments. Bar graphs in (b, d-g) represent means + s.e.m. Stain-Free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Western Blot, Over Expression, Infection, Control, Expressing, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Experimental setup for identification of SBPs in a SILAC-based approach. pacSph treatment of Sgpl1 ΔIns1E and CerS2:Sgpl1 ΔIns1E cells differentially labelled with stable isotopes allows crosslinking of SL-protein complexes by UV irradiation (with omission of UV irradiation as a control condition), followed by cell lysis and conjugation of biotin to the SL-protein complexes. After Streptavidin-based pull-down, SBPs can be identified and quantified in the same MS run by the differing peptide mass due to SILAC isotope labelling. b , Volcano plot showing log 2 fold change of proteins pulled down from pacSph-treated Sgpl1 ΔIns1E (+UV) versus Sgpl1 ΔIns1E (−UV) cells plotted against the −log 10 P values of a one-sample two-sided t -test against 0. Proteins with log 2 fold change >1 and a BH-FDR <0.05 are regarded as SBPs ( n = 4 independent experiments). c , Volcano plot showing log 2 fold change of SBPs identified in b (Supplementary Fig. ) and pulled down from pacSph-treated CerS2:Sgpl1 ΔIns1E (+UV) versus Sgpl1 ΔIns1E (+UV) cells plotted against the −log 10 P values of a two-sample two-sided equal variance t -test ( n = 4 independent experiments). SBPs with a fold change >1.5 and a BH-FDR <0.05 were regarded as Cers2-dependent SBPs. Fold enrichment was 6.55 and FDR-corrected P value was 3.12 −10 for GO term ‘endoplasmic reticulum’. d , pacSph pull-down of endogenous Tmed2 in Sgpl ΔIns1E and Sgpl1:CerS2 ΔIns1E cells ( n = 4 independent experiments); exemplary immunoblot (right) and quantification (left). Eluate intensities were normalized to respective input intensities. e , Relative mRNA expression of Tmed1, Tmed2 and Pcsk1 in murine pseudoislets transfected with control siRNA or siRNA against Tmed1, Tmed2 or both. n = 4 independent experiments. f – i , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in pseudoislets transfected with siRNA as described in Extended Data Fig. ; n = 3 independent experiments. f , Representative immunoblot. g , Quantification of Pcsk1. h , Quantification of Pro-Pcsk1. i , Ratio of Pcsk1 to Pro-Pcsk1. j – l , Insulin content ( j ), proinsulin content ( k ) and ratio of insulin to proinsulin ( l ) in pseudoislets transfected with siRNA as described in Extended Data Fig. determined via ELISA ( n = 8 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test ( d ) and repeated measures one-way ANOVA with Tukey’s multiple comparisons test ( e and g – l ). In e , ANOVA was performed for each mRNA target individually. P values are stated in each figure. Data points in d , e and g – l represent individual experiments. Bar graphs represent mean ± s.e.m. For one experiment in j – l , the mean of five replicates, consisting of nine pseudoislets, respectively, was plotted per condition. Stain-free signal was used for normalization of immunoblots in g – i . Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Multiplex sample analysis, Irradiation, Control, Lysis, Conjugation Assay, Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Top 20 SBPs identified in Fig. according to p -values and a log 2 fold change > 3. b , Verification of Bst2 and Fxyd6 as SBPs by overexpression of DDK-tagged variants in Sgpl1 ΔIns1E cells followed by pacSph-pulldown; representative immunoblots (left) and quantification (right). Eluate band intensities were normalized to input bands and +UV samples were set to 1 ( n = 4 independent experiments). c , Verification of Tmed1 as SBP as described in (b); representative immunoblot (left) and quantification (right). n = 3 independent experiments. d , Immunoblot detection of Tmed2 protein levels in Sgpl1 ΔIns1E and Cers2:Sgpl1 ΔIns1E cells. Representative immunoblot showing 3 replicates per genotype (left) and quantification (right). n = 3 independent experiments with 3 replicates per genotype, respectively. e , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 Cers2 ΔBKO mice. Representative immunoblot (left) and quantification (right). f , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 ob/ob.B6 mice at week 12. Representative immunoblot (left) and quantification (right). g , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 db/db.BKS mice at week 12. Representative immunoblot (left) and quantification (right). Statistical analysis was performed using a one sample t -test against 1 (b, c) or Student’s t -test (d-g). P -values are stated in each figure. Bar graphs represent means + s.e.m. Data points in (b, c and d) represent individual experiments. Data points in (e-g) represent islets from individual mice. Stain-Free signal was used for normalization of all immunoblots, except for (b) and (c). Stain-Free images of (e), (f) and (g) were reproduced from Fig. , Extended Data Fig. , as the same PVDF membranes were used for detection of Pro-Pcsk1, Pcsk1 and Tmed2, respectively. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Over Expression, Western Blot, Control, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Double cut CrispR/Cas9 knockout strategy for Tmed2 in Ins1E cells. b, c , Relative mRNA expression of various Tmed family members (b) and beta cell identity markers (c) in wildtype Ins1E, control and Tmed2 ΔIns1E cells. Wildtype Ins1E samples were set to 1 (dotted line). n = 3 independent experiments. Note that potentially as a sign of attempted compensation, Pcsk1 mRNA levels are increased in Tmed2-deficient Ins1E cells. d , Representative immunoblot analysis of Tmed2 protein expression in 3 control vs. 3 Tmed2 ΔIns1E replicate lysates. e , Insulin content in control and Tmed2 ΔIns1E cells determined via ELISA. n = 3 independent experiments with 3 replicates per genotype, respectively. Statistical analysis was performed using multiple two-sided t -tests with Holm-Sidak correction (b and c) and a two-sided Student’s t -test (e). Data points represent independent experiments. Bar graphs represent means + s.e.m. The control and Tmed2 ΔIns1E cells are pools of individual monoclonal cell lines, respectively (8 monoclonal control cell lines and 3 monoclonal Tmed2 ΔIns1E cell lines were used for pooling). Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: CRISPR, Knock-Out, Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Co-immunoprecipitation (Co-IP) of co-overexpressed Tmed2-V5 and Pro-Pcsk1/Pcsk1-DDK in Ins1E cells. Representative immunoblot (left) and quantification of three replicate experiments (right). As Ctrl-plasmid, the promotorless pNL1.3 from Promega (N1021) was used. b , Representative confocal images for co-localization of overexpressed Tmed2-V5 and Pro-/Pcsk1-DDK in Ins1E cells. Green, SytoxGreen as nucleus marker; red, Pro-/Pcsk1-DDK; blue, Tmed2-V5. Scale bar, 5 µm. c, d , Quantification of overlap of Pro-/Pcsk1-DDK with Tmed2-V5 (c) and Tmed2-V5 with Pro-/Pcsk1-DDK (d) in control and CerS2 ΔIns1E cells. n = 2 independent experiments; only one experiment shown. e-j , Overlap of ER-marker PDI and Golgi-Marker TGN46 with Tmed2-V5, Pro-/Pcsk1-DDK (allowing detection of both Pro-Pcsk1 as well as mature Pcsk1) and Pro-Pcsk1 (only allowing detection of the immature Pro-Pcsk1 protein) after overexpression in control and CerS2 ΔIns1E cells. n = 3 independent experiments. Statistical analysis was performed using a paired two-sided Student’s t -test (a) and unpaired two-sided Students t -tests (c-j). Data points represent replicate experiments (a) and individually quantified cells (c-d) or well sites (e-j). Bar graphs represent means + s.e.m. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Marker, Control, Over Expression