Figure 2.
Functional evaluation of predicted potentially deleterious homozygous missense OSMR variants from the general population (overexpressed in OSMR- and LIFR-deficient HEK293T cells). (A) All homozygous OSMR variants from gnomAD v4.1 plotted as CADD score versus MAF. The red dot refers to the variant of P1. Dotted line represents the MSC (>19.3) with 95% confidence interval. A total of 11 homozygous missense OSMR variants predicted to be potentially deleterious (CADD score >20) from gnomAD v4.1 are listed in the table with the variant identified in P1 shown in red. (B) Localization of the predicted deleterious OSMR variants in different domains of the OSMR protein with the variant in P1 shown in red. CBD, cytokine binding domain. (C) The surface receptors OSMR and LIFR known to bind OSM were knocked out in HEK293 cells by the CRISPR/Cas9 technique. KO efficiencies of two SCCs were determined by EditCo’s ICE Analysis Tool (52) and shown as indels. (D and E) Functional validation of nucleofected HEK293T cells with CRISPR guides targeting AAVS1 (control) or OSMR and LIFR from C. Cells were stimulated for 30 min by either OSM (50 ng/ml), IL-6 (100 ng/ml), or IL-11 (100 ng/ml) prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in E. (F and G) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or various OSMR variants from gnomAD by transfection. Cells were left untreated (UT) or stimulated with 50 ng/ml OSM for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, OSMR, GFP, and GAPDH, with quantification of WB band intensities shown in G. (H and I) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or the OSMR P1 variant V436D by transfection. Cells were left untreated (UT) or stimulated with OSM (50 ng/ml), IL-6 (100 ng/ml), IL-11 (100 ng/ml), or IL-31 (100 ng/ml) for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in I. Source data are available for this figure: SourceData F2.

Functional evaluation of predicted potentially deleterious homozygous missense OSMR variants from the general population (overexpressed in OSMR- and LIFR-deficient HEK293T cells). (A) All homozygous OSMR variants from gnomAD v4.1 plotted as CADD score versus MAF. The red dot refers to the variant of P1. Dotted line represents the MSC (>19.3) with 95% confidence interval. A total of 11 homozygous missense OSMR variants predicted to be potentially deleterious (CADD score >20) from gnomAD v4.1 are listed in the table with the variant identified in P1 shown in red. (B) Localization of the predicted deleterious OSMR variants in different domains of the OSMR protein with the variant in P1 shown in red. CBD, cytokine binding domain. (C) The surface receptors OSMR and LIFR known to bind OSM were knocked out in HEK293 cells by the CRISPR/Cas9 technique. KO efficiencies of two SCCs were determined by EditCo’s ICE Analysis Tool (52) and shown as indels. (D and E) Functional validation of nucleofected HEK293T cells with CRISPR guides targeting AAVS1 (control) or OSMR and LIFR from C. Cells were stimulated for 30 min by either OSM (50 ng/ml), IL-6 (100 ng/ml), or IL-11 (100 ng/ml) prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in E. (F and G) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or various OSMR variants from gnomAD by transfection. Cells were left untreated (UT) or stimulated with 50 ng/ml OSM for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, OSMR, GFP, and GAPDH, with quantification of WB band intensities shown in G. (H and I) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or the OSMR P1 variant V436D by transfection. Cells were left untreated (UT) or stimulated with OSM (50 ng/ml), IL-6 (100 ng/ml), IL-11 (100 ng/ml), or IL-31 (100 ng/ml) for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in I. Source data are available for this figure: SourceData F2.

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