Figure 3.
OSMR LOF variants lead to a lack of OSMRβ cell surface expression in HEK293 cells. (A) Extracellular and total OSMRβ expression were quantified in HEK293 cells transfected with WT OSMRβ, OSMRβ missense variants, (p.Ala349Asp and p.Val436Asp), or FPLCA variants (p.Asn462Ser, p.Gly513Asp, p.Val631Leu, and p.Ile691Thr) using flow cytometry. OSMRβ expression was quantified in GFP+ cells; n = 3. (B) Extracellular and total OSMRβ expression were quantified in HEK293 cells transfected with either WT OSMRβ or OSMRβ variants that lead to a frameshift and premature stop (p.Gln51Thrfs*23, p.Gln270*, p.Pro478Hisfs*18, and p.Tyr660Serfs*16) using flow cytometry. OSMRβ expression was quantified in PE-OSMRβ+ cells instead of GFP+ cells as the variant results in a truncated protein, preventing translation of the C-terminal GFP; n = 3. (C) Quantification of A, extracellular OSMRβ expression; n = 3. (D) Quantification of A, total OSMRβ expression; n = 3. (E) Quantification of B, extracellular OSMRβ expression; n = 3. (F) Quantification of B, total OSMRβ expression; n = 3. (G) Immunoblot visualizing OSMRβ expression in HEK293 cells transfected with WT OSMRβ or the following OSMRβ variants, p.Gln51Thrfs*23, p.Gln270*, p.Pro478Hisfs*18, and p.Tyr660Serfs*16; n = 3. Statistical comparisons between OSMRβ WT and OSMRβ variants were performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Asterisks indicate statistical significance (P < 0.05). Source data are available for this figure: SourceData F3. Refer to the image caption for details. Panel A shows two sets of histograms comparing extracellular and total OSMR expression in HEK293 cells transfected with wild-type OSMR, missense variants, and FPLCA variants. The x-axis represents OSMR expression levels, while the y-axis indicates the frequency of cells. Panel B presents similar histograms for wild-type OSMR and premature stop variants. Panel C is a bar graph quantifying extracellular OSMR expression for missense variants, with the y-axis showing MFI fold change relative to the empty vector. Panel D is a bar graph quantifying total OSMR expression for missense variants, with the same y-axis as Panel C. Panel E quantifies extracellular OSMR expression for premature stop variants, and Panel F quantifies total OSMR expression for these variants. Panel G is an immunoblot visualizing OSMR expression in cells transfected with wild-type OSMR and premature stop variants, with beta-actin as a loading control.

OSMR LOF variants lead to a lack of OSMRβ cell surface expression in HEK293 cells. (A) Extracellular and total OSMRβ expression were quantified in HEK293 cells transfected with WT OSMRβ, OSMRβ missense variants, (p.Ala349Asp and p.Val436Asp), or FPLCA variants (p.Asn462Ser, p.Gly513Asp, p.Val631Leu, and p.Ile691Thr) using flow cytometry. OSMRβ expression was quantified in GFP+ cells; n = 3. (B) Extracellular and total OSMRβ expression were quantified in HEK293 cells transfected with either WT OSMRβ or OSMRβ variants that lead to a frameshift and premature stop (p.Gln51Thrfs*23, p.Gln270*, p.Pro478Hisfs*18, and p.Tyr660Serfs*16) using flow cytometry. OSMRβ expression was quantified in PE-OSMRβ+ cells instead of GFP+ cells as the variant results in a truncated protein, preventing translation of the C-terminal GFP; n = 3. (C) Quantification of A, extracellular OSMRβ expression; n = 3. (D) Quantification of A, total OSMRβ expression; n = 3. (E) Quantification of B, extracellular OSMRβ expression; n = 3. (F) Quantification of B, total OSMRβ expression; n = 3. (G) Immunoblot visualizing OSMRβ expression in HEK293 cells transfected with WT OSMRβ or the following OSMRβ variants, p.Gln51Thrfs*23, p.Gln270*, p.Pro478Hisfs*18, and p.Tyr660Serfs*16; n = 3. Statistical comparisons between OSMRβ WT and OSMRβ variants were performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Asterisks indicate statistical significance (P < 0.05). Source data are available for this figure: SourceData F3.

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