Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: ChIP-sequencing (ChIP-seq) against Irx3 was performed in preadipocytes from iWAT one day before (day -1) and one day after (day 1) initiation of differentiation, n = 2, as well as from gWAT on day 1, n = 1. A) Number of Irx3 ChIP-seq peaks for each condition shown as a Venn diagram. Only high-confidence (HC) peaks with q-value below 10e-4 and enrichment above 10X were included. B) Distribution of Irx3 ChIP-seq peaks across the genome (top) and relative to nearest TSS (bottom). C) Enriched REACTOME pathway ontology terms for Irx3-bound genes. See also Supplementary file 1 for Panther GOs. D) Genes in the enriched GO category related to Sumoylation. E) Genome browser tracks for each ChIP-seq conditions at the loci of the Sumo-related genes. F) Genes in the enriched GO category related to Chromatin modifying enzymes.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Open chromatin profiling was performed by ATAC-sequencing at seven timepoints during differentiation of preadipocytes derived from gWAT of male and female mice. A) Total number of ATAC peaks shown for each timepoint during gWAT differentiation. B) Heatmap of the ATAC peaks that displayed significant time-dependent change in magnitude. Normalized row z-scores shown. C) Clustering of time-dependent ATAC peaks according to maSigPro stepwise regression. The total number of ATAC peaks, as well as the number of Irx3-bound ATAC peaks shown. D) Selected Panther GOs of the two clusters comprising loci with increased chromatin opening during differentiation. Fold enrichment and FDR shown. See also Supplementary file 2 for all GOs. E) Heatmap comparing enrichment of ChIP-seq peaks from all conditions (iWAT and gWAT) with ATAC-seq peaks in gWAT day 1. F) Percentage of ChIP-seq peaks overlapping ATAC-seq peaks for each timepoint during differentiation. Asterisks denote the timepoints where ChIP-seq was performed and thereby are directly comparable with ATAC-status. Color of bars represent degree of ATAC-enrichment/chromatin openness. G) Percentage of Irx3-bound ATAC peaks displaying changes over time (left). Distribution of Irx3-bound, time-dependent ATAC peaks according to the six global ATAC clusters (right). Enrichment of time-dependent Irx3-bound versus total ATAC-seq peaks shown (bottom). H) Heatmap illustrating the high proportion of Irx3-bound, time-dependent ATAC peaks that belong to cluster 4 and 6; loci that display increased opening in mature adipocytes. Normalized row z-scores shown. See (B) for corresponding heatmap of total ATAC peaks

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Sequencing, Derivative Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Open chromatin profiling was performed by ATAC-sequencing.at three timepoints during differentiation of beige ME3 preadipocytes A) Total number of ATAC peaks shown for each timepoint during ME3 differentiation. B) Heatmap of the ATAC peaks that displayed significant time-dependent change in magnitude. Normalized row z-scores shown. C) Clustering of time-dependent ATAC peaks in ME3 according to maSigPro stepwise regression. The total number of ATAC peaks, as well as the number of Irx3-bound ATAC peaks shown. D) Selected Panther GOs of cluster 2, comprising loci with increased chromatin opening during differentiation. Fold enrichment and FDR shown. See also Supplementary file 2 for all GOs. E) Heatmap comparing enrichment of ChIP-seq peaks from all conditions (iWAT and gWAT) with ATAC-seq peaks in ME3 days 0, 1 and 7. F) Percentage of Irx3-bound ME3 ATAC peaks displaying changes over time (top). Distribution of Irx3-bound, time-dependent ATAC peaks in the six global ME3 ATAC clusters (middle). Enrichment of time-dependent Irx3-bound versus total ATAC-seq peaks shown (bottom). G) Heatmap illustrating the high proportion of Irx3-bound, time-dependent ME3 ATAC peaks that belong to cluster 2; loci that display increased opening in mature adipocytes. See (B) for corresponding heatmap of total ME3 ATAC peaks. H) Panther GOs for genes bound by Irx3 and displaying increased chromatin opening during ME3 differentiation. I) Genome browser aligning ChIP-seq tracks with ATAC-seq tracks from all timepoints in ME3 cells, gWAT cells on the promoter of the Kdm3a gene.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Sequencing, ChIP-sequencing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) Venn diagram of Irx3-bound genes and differentially expressed genes in response to Irx3- KO. The size of each circle is not drawn according to scale. B) Heatmap displaying Irx3 ChIP-seq peak enrichment and the direction and magnitude of each corresponding Irx3-bound gene in response to Irx3-KO on days 1 and 7 of adipocyte differentiation. C) Enriched Panther and Reactome ontology terms for the 122 Irx3 target genes with differential expression on day 1 of differentiation in response to Irx3- KO. D) Genes constituting the GO categories “H3K4 mono/demethylation” on day 1 of differentiation. Log 2 -fold changes for each gene shown. E) Genes constituting the GO category “Chromosome organization” on day 1 of differentiation. Log 2 -fold changes for each gene shown. F) Enriched Panther and Reactome ontology terms for the 179 Irx3 target genes with differential expression on day 7 of differentiation in response to Irx3- KO. G) Genes constituting the GO categories “Chromatin modifying enzymes” on day 7 of differentiation. H) Genes constituting the GO categories “Nucleosome organization” on day 7 of differentiation. I) Genes constituting the GO categories “Histone H4 acetylation” on day 7 of differentiation. J) Genes constituting the GO categories “Histone H2A acetylation” on day 7 of differentiation. Log 2 -fold changes for each gene shown. Downregulated genes, blue bars; upregulated genes, red bars. See for fold change of genes in the remaining categories.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Log2-fold changes for each gene in Irx3 -KO vs control cells for each GO category is shown. Downregulated genes, blue bars; upregulated genes, red bars. Related to .

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques:

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) Irx3 ChIP-seq peaks at the promoter (TSS ±1.5 kb) of key genes involved in sumoylation one day before and/or one day after initiation of adipogenic differentiation in preadipocytes isolated from iWAT and gWAT (top). ATAC-seq at the same loci in ME3 control and Irx3- KO cells on day 1 of adipogenic differentiation (middle). Differences in expression of the respective genes between ME3 control and Irx3- KO cells on day 1. Log 2 -fold changes and fold changes shown (bottom). B) Overview of the sumoylation cycle. The genes shown in (A) are highlighted in red letters with arrows indicating direction of change with Irx3- KO. The Sumo genes encode prepeptides that can be cleaved and activated by Senp proteases to reveal a GG diglycine motif (1). The GG motif in free, mature SUMO forms a thioester bond with the E1 activating complex, consisting of the Sae1 and Uba2 (also known as Sae2) heterodimer (2). The SUMO group is next transferred to the E2 conjugating enzyme Ubc9 (also known as Ube2i) (3), which in turn transfers the SUMO group to the target protein with the aid of E3 ligases (4-5). Multiple SUMO subunits can be attached together to form a poly-SUMO tail (6). SUMO groups can be released from target proteins by Senp or other SUMOylase family members (7). C) Western blot showing global SUMOylome pattern on days 0 and 1 of adipogenic differentiation in ME3 control and Irx3- KO cells. D) Volumetric quantification of bands in (C). Levels of conjugated SUMO relative to β-Actin shown. * p < 0.05; ns, not significant, two-way ANOVA with Holm-Sidak correction for multiple testing. E) Western blot showing global SUMOylome pattern on days 0 and 1 of adipogenic differentiation in ME3 control and Irx3- KO cells transfected with either empty plasmid or AM-tagged Irx3. F) Volumetric quantification of bands in (E). Relative levels of conjugated SUMOylome relative to GAPDH shown.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: ChIP-sequencing, Isolation, Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) ME3 control and Irx3- KO cells were treated with either vehicle or 0.5 µM of the sumoylation inhibitor ML-792 from days-2 to 9 of adipogenic differentiation. On day 9, the cells were treated with the Bodipy fluorescent lipid stain for 1 hour and imaged with fluorescence and brightfield microscopy. Representative images from 9 images/well in biological triplicates, from one of four independent experiments. Scale bar = 400 µm. See for all images from this experiment. B) Magnification of the images shown in (A). Scale bar = 50 µm. C) Quantification of Bodipy total integrated intensity, number of lipid droplet and average lipid volumes. Data from all 9 images/well were merged to represent each well. Three replicate wells from one of four independent experiments are shown. Data normalized to relative cell count. ***p < 0.001, control vs Irx3- KO cells; ## p <0.01, ### p < 0.001, ML-792 vs DMSO; ns, not significant, two-way ANOVA with Holm-Sidak correction for multiple testing. D) Gene expression in ME3 control and Irx3- KO cells in response to 0.5 µM ML-792, same cells as (A-C). Ratios between ML-792 and DMSO for control and Irx3- KO cells shown for simplicity. Rps13 was used as reference gene. See for full gene expression data. *p < 0.05, **p < 0.01, multiple Student’s t-tests with Holm-Sidak correction for multiple testing. E) Dose-response effect of ML-792 on lipid accumulation in Irx3- KO cells when ML-792 was added on either day -2 or 0 and kept throughout differentiation until day 9. Data from 4 images/well merged to represent each well. Quantification of four replicate wells from one of three independent experiments shown. See for images. *p < 0.05, ***p <0.001, ### p < 0.001; ns, not significant, two-way ANOVA with Holm-Sidak correction for multiple testing. F) The effect of keeping 0.5 µM ML-792 at different stages of differentiation (blue bars), or only during various 2-day intervals (red bars). Data normalized to relative cell count and shown as ML-792/DMSO ratio. ***p < 0.001, one-way ANOVA with Holm-Sidak correction for multiple testing. G) Schematic with proposed mechanism for Irx3 and Sumo-mediated regulation of adipogenesis.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Staining, Fluorescence, Microscopy, Cell Counting, Expressing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) ME3 control and Irx3- KO cells were treated with either vehicle or the SUMOylation inhibitor ML-792 from days -2 to 9 of adipogenic differentiation. On day 9, the cells were treated with the Bodipy fluorescent lipid stain for 1 hour and imaged with fluorescence microscopy. Nine images/well were stitched to represent each well of a 24-well plate. Bodipy signal in triplicate wells from one of four independent experiments is shown. Scale bar = 400 µm. B) Gene expression in ME3 control and Irx3- KO cells in response to 0.5 µM ML-792, relative to Rps13 . *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Holm-Sidak correction for multiple testing. C) Dose-response effect of ML-792 on lipid accumulation in Irx3- KO cells when ML-792 was added on either day -2 or 0 and kept throughout differentiation until day 9. Four images from each well of a 96-well plate were stitched together. One representative well from one of three independent experiments is shown with Bodipy fluorescent signal (left), brightfield (middle) and merge (right). D) Dose-response effect of ML-792 on relative cell count in Irx3- KO cells. n = 8 replicate wells from one of three independent experiments. Related to .

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Staining, Fluorescence, Microscopy, Expressing, Cell Counting

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) Differential expression of adipogenic regulators in Irx3- KO vs control cells. Experimentally validated binding of upstream transcription factors to the promoters of each adipogenic regulator in WT cells is shown to the right, ChIP-seq data collected from the UCSC genome browser hub UniBind 2021 . Transcription factors experimentally shown to be sumoylated in WT preadipocytes or mature adipocytes are marked with (S), data from . B) Luciferase activity of a reporter construct under control of 3xPPRE sites co-transfected with Pparg and/or Pgc-1a. Firefly luciferase units relative to the control group and normalized to constitutive Renilla luciferase is shown. Data show mean ± SD of n= 3 replicates from one out of two independent experiments.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Expressing, Binding Assay, ChIP-sequencing, Luciferase, Activity Assay, Construct, Transfection

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Global mRNA-sequencing was performed to map differentially expressed genes (DEGs) between ME3 control and Irx3- KO cells on day 1 of differentiation. This list was compared with either differences in newly transcribed genes (DNTs) as measured by SLAM-seq (A-C), or DEGs as measured by mRNA-seq (D-F) in response to ML-792 in 3T3-L1 cells. A) Venn diagram showing overlap between Irx3- KO mRNA-seq DEGs and ML-792 SLAM-seq DNTs on day 1 of differentiation. B) Heatmap showing direction of change among overlapping genes in (A). C) Selection of top enriched/most significant GO categories among the overlapping genes in (A-B). See and Supplementary file 4 for complete lists. D) Venn diagram showing overlap between mRNA-seq DEGs in response to Irx3- KO or ML-792 on day 1 of differentiation. E) Heatmap showing direction of change among overlapping genes in (D). F) Selection of top enriched/most significant GO categories among the overlapping genes in (D-E). See and Supplementary file 5 for complete lists. G) A pooled list of genes responding to ML-792 identified by SLAM-seq and mRNA-seq was overlapped with a SUMO-ChIP-seq dataset to make a list of direct SUMO target genes (Supplementary file 6), which in turn was compared with the list of Irx3-responsive genes. H) Selection of top enriched/most significant GO categories among the overlapping genes in (G). See Supplementary file 6 for complete lists.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Sequencing, Selection, ChIP-sequencing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Global mRNA-sequencing was performed to detect differentially expressed genes (DEGs) between ME3 control and Irx3- KO cells on day 1 of differentiation, using FC ≥1.2 and padj. ≤0.001. SLAM-seq was performed to detect changes in newly transcribed genes between 3T3-L1 cells treated with vehicle or 0.5 µM ML-792 on day 1 of differentiation, using FC ≥1.2 and padj. ≤0.05. Overlapping DEGs were subjected to combined GO analyses from Panther Pathways, Panther Biological processes complete and Reactome pathways. Top enriched/most significant categories are shown. Related to . For complete lists, see Supplementary file 4. A) Top GOs for upregulated genes in both datasets. B) Top GOs for downregulated genes in both datasets. C) Top GOs for genes up- and downregulated with Irx3- KO and ML-792, respectively. D) Top GOs for genes down- and upregulated with Irx3- KO and ML-792, respectively.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Global mRNA-sequencing was performed to detect differentially expressed genes (DEGs) between ME3 control and Irx3- KO cells on day 7 of differentiation, using FC ≥1.2 and padj. ≤0.001 and DEGs between 3T3-L1 cells treated with vehicle or 0.5 µM ML-792 on day 7 of differentiation, using FC ≥1.2 and padj. ≤0.001. A) Venn diagram showing overlap between DEGs following Irx3- KO or ML-792 on day 7 of differentiation. B) Heatmap showing direction of change among overlapping genes in (A). C) Selection of top enriched/most significant GO categories among the overlapping genes in (A-B).

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Sequencing, Selection

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) Open chromatin profiling was performed by ATAC-seq in control and Irx3- KO cells on day 0 of differentiation. Selected top GO for more open peaks in promoters of Irx3- KO cells shown. See also . B) UCSC genome browser tracks showing examples ATAC-seq peaks from control and Irx3- KO cells at the promoters of genes from two osteoblast-related GO categories in (A). C) ME3 control cells subjected to adipogenic differentiation in 3D culture. I, brightfield view of Oil-red-O lipid-stained cells. II, phase contrast view of Oil-red-O staining. III, fluorescent view of the hydrogel surface with DAPI nuclear stain (blue), Bodipy lipid stain (green) and F-actin cytoskeletal stain (red). IV, fluorescent view of the inside of the hydrogel. V and VI, zoomed in fluorescent view. D) Irx3- KO control cells subjected to adipogenic differentiation in 3D culture. I-V, same conditions as in C. E) ME3 control and Irx3- KO cells treated with growth medium (GM) or osteogenic medium (OM), followed by Alkaline Phosphatase (ALP) staining. Brightfield view (left panel) and quantification (right panel) of ALP staining. F) ME3 control and Irx3- KO cells treated with growth medium (GM) or osteogenic medium (OM), followed by Alizarin Red S staining (ARS). Brightfield view (left panel) and quantification (right panel) of the ARS staining. G) Expression of early, intermediate and late osteogenic markers in in control and Irx3- KO cells in response to differentiation in growth medium or osteogenic medium. Two-way Anova with Holm-Sidak correction for multiple testing. # p adj < 0.05, ### p adj < 0.001, osteogenic vs growth medium. * p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001 control vs Irx3- KO. NS, not significant.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: ME3 control and Irx3- KO cells were treated with adipogenic stimuli for 7 days, and open chromatin profiling on promoters was performed by ATAC-seq on days 0, 1 and 7. Most enriched and/or significant GOs for more open chromatin on day 0 (A), day 1 (C) and day 7 (E) and less open chromatin on day 0 (B), day 1 (D) and day 7 (F) shown. See supplementary file 8 for complete lists.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques:

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: Global changes in open chromatin in promoters of Irx3- KO vs control ME3 cells was measured by ATAC-seq and compared with changes in gene expression by RNA-seq. Log2-fold changes on day 1 (left) and day 7 (right) is shown.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

doi: 10.1101/2023.10.17.562662

Figure Lengend Snippet: A) ME3 control and Irx3- KO cells were subjected to osteogenic differentiation in 3D culture and treated with either vehicle or 0.5 µM ML-792. On day 21, the cells were stained with alizarin Red S. Brightfield view (left panel) and quantification (right panel) shown. Data represent n=5 replicate wells from a single experiment. B) Cells were treated as in (A), except RNA was harvested on day 7 and expression of early and intermediate markers of osteogenesis was measured by qPCR. Data shown relative to the reference gene Tbp and the control group. Data represent n=5 replicate wells from a single experiment. Two-way Anova with Holm-Sidak correction for multiple testing. # p adj < 0.05, ### p adj < 0.001, control vs Irx3- KO. * p adj < 0.05, *** p adj < 0.001 DMSO vs ML-792. Nns, not significant.

Article Snippet: Briefly, mouse Irx3 (NM_008393) was amplified by PCR from the donor vector pCMV6-entry-Irx3-myc-DDK (cat. no MR208149, Origene, Rockville, MD, USA) with the following forward 5’- CGATCTAAGTAAGCTT CACC ATGTCCTTCCCCCAGCTCG-3’ and reverse 5’- GATCTTGGCAAAGCT TAGACGAGGAGAGAGCTGATAAGACC-3’ primers.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) CRISPR/Cas9 genome editing and PCR genotyping of LAMP2A KO. (B) qRT-PCR analysis of LAMP1, LAMP2A, LAMP2B and LAMP2C expression in WT and KO cells from HT1080 and A549 cell lines (C) Western blot analysis of LAMP2A and LAMP1 in WT and KO (D) Confocal analysis of immunofluorescence with anti-LAMP2A (Green) and anti-LAMP1 (Red) antibodies in WT and KO cells from HT1080 and A549 cell lines. DAPI was used to counterstain cell nuclei. The scale bar (in white) is 20 µm. Insets at higher magnification as indicated. (E) Western blot analysis of LAMP2A, LAMP1 and LAMP2B in WT and KO xenograft tumors from HT1080 and A549 cell lines. Immunofluorescence staining for (F) LAMP2A or (G) ID1 in WT and KO HT1080 or A549 tumors. Scale bar is100 μm. Insets at higher magnification as indicated. Quantification of the ID1+ cell ratio per 20× field (bar graph). (H) Western blot analysis of ID1 in WT and KO xenograft tumors from HT1080 cell lines. Error bars, ±SD. ***p<0.001; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: CRISPR, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) proliferation (B) clonogenic growth (C) experimental overview of LAMP2KO implantation of 4 tumors (2 KO vs. 2 WT) per mice. (D) Immunohistochemical staining for LAMP2A and LAMP1 in WT and KO tumors from HT1080 and A549 cell lines (n=3 mice). The scale bar (in black) is 50 µm. Insets at higher magnification as indicated. (E) Representative images of the xenograft mice and tumors. (F and G) Tumor growth and weight (n=3 mice). (H) Representative image of EdU positive (EdU+, Green) and DAPI-(blue) labeled HT1080 WT and KO tumor slides (n=3). Scale bar is 100 μm (H, left). Quantification of the EdU+ cell ratio per 20× field (bar graph). Error bars, ±SD. ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Immunohistochemical staining, Staining, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) The transcript abundance of LAMP2A in three non-neoplastic human lung tissues compared to 8 primary human lung tumors. (B) LAMP2A expression in human metastatic and primary cancer tissues (n=5, for each patient). (C) Schematic picture of human NSCLC primary tumors and brain metastasis in cancer patient. (D) Comparative RNA expression analysis of human NSCLC primary tumors to match brain metastasis for EMT markers. (E) Tissue-Microarray analysis (TMA) of the LAMP2A and Vimentin protein expression level in 184 human multiorgan metastasis array. Two representative cores are shown in the figure to illustrate the inverse LAMP2A/Vimentin staining patterns. Scale bar 50 µm. (F) table revealing the number of patients with inverse expression of LAMP2A/Vimentin and its association with tumor grade. Error bars, ±SD. **p<0.01, ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Expressing, RNA Expression, Microarray, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) Western blot analysis of three anti-LAMP2A antibodies in A549 WT and KO cells. (B) The immunostaining with three anti-LAMP2A antibodies in A549 WT and KO cells (red). DAPI was used to counterstain cell nuclei (Blue). The scale bar (in white) is 5 µm. Insets at higher magnification as indicated. (C) Immunohistochemical staining for LAMP2A in A549 WT and KO tumors by three LAMP2A antibodies (n = 3-4 mice). Red arrows mark the area of staining in the mouse stroma (tumor-supporting mice tissues). The scale bar (in black) is 50 µm. Insets at higher magnification as indicated.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Western Blot, Immunostaining, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) qPCR analysis of LAMP2 expression in the indicated set of cell lines. (B) The effect of TGFβ treatment on VIM, CDH1 and LAMP2A mRNA. The effect of TGFβ signaling inhibition (by Tranilast) or CMA activation on LAMP2A mRNA in OVPA8 cells. (C) The effect of CMA activation on TGFβR2 protein level in the ES2 cell line. (D) The effect of siRNA-mediated knockdown by two independent siRNAs targeting TGFβR2 on LAMP2A in FU-OV1 cells (left panel) and ES2 cells (right panel). (E) The effect of CMA activation, Tranilast, Tranilast +chloroquineon and CMA +chloroquineon treatment on TGFβR2. (F) The effect of LAMP2A knockdown on the TGFβR2 lysosomal enrichment as analyzed by mass spectrometry. (G) The effect of siRNA-mediated knockdown by two independent siRNAs targeting LAMP2A, on TGFβR2. (H) The effect of re-expression (RE) of LAMP2A in the LAMP2A KO HT1080 cells. Error bars, ±SD. **p<0.01, ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Expressing, Inhibition, Activation Assay, Mass Spectrometry, Two Tailed Test

Journal: bioRxiv

Article Title: Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

doi: 10.1101/381541

Figure Lengend Snippet: Equal cell numbers were seeded 48 hours before confluence measurement using live cell imaging on IncuCyte Zoom (Essen BioScience). In case of transient transfection with PGRMC2-GFP and GFP-PGRMC1 the proliferation of fluorescent cells was measured which starts at cell density of about zero. Growth curve analysis and growth constant k (slope of regression curve) calculation was done using multilevel regression technique using R Studio.

Article Snippet: The plasmids for transient transfections pEGFP-C1-PGRMC1 and pCMV6-AC-PGRMC2-GFP vector (RG204682) (OriGene Technologies, Rockville MD, USA) were used at a concentration of 0.01 μg/μl at an optimized transfection ratio of 1:4 diluted in pure DMEM/F12.

Techniques: Live Cell Imaging, Transfection

Journal: bioRxiv

Article Title: Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

doi: 10.1101/381541

Figure Lengend Snippet: ( A ) Human adrenocortical NCI-H295R cells at different cell division stages were stained with anti-PGRMC1 (green), anti-Aurora kinase A (AURKA) (red) and DAPI (blue). ( B ) Human adrenocortical NCI-H295R cells at meta- and telophase were stained with anti-PGRMC1 (green), anti-α-tubulin (TUBA) (red), anti-β-tubulin (TUB) (red), anti-PGRMC2 (red), anti-Aurora kinase B (AURKB) (red) and DAPI (blue). Scale bars: 5 μm. ( C ) Schematic drawing of cellular localization of ALADIN, Aurora kinase A, PGRMC1 and PGRMC2 during mitosis. MT, microtubule.

Article Snippet: The plasmids for transient transfections pEGFP-C1-PGRMC1 and pCMV6-AC-PGRMC2-GFP vector (RG204682) (OriGene Technologies, Rockville MD, USA) were used at a concentration of 0.01 μg/μl at an optimized transfection ratio of 1:4 diluted in pure DMEM/F12.

Techniques: Staining