PI3K subunit expression and signalling in primary dermal fibroblasts.

Immunoblotting of AKT, AKT phosphorylated at threonine 308 (T308) or serine 473 (S473), p85α, p110δ and p110α and are shown with and without stimulation by 100 nM insulin (Ins) for 10 minutes. β-actin is shown as a loading control, with different amounts of pooled lysate (Pool) used to demonstrate signal intensity in the linear range. Results from 4 healthy controls (WT; 1-4), 1 patient with Activating p110 Delta Syndrome 2 (APDS2) due to the p85α Δexon11 variant, and 3 patients with PIK3CA-Related Overgrowth Spectrum (PROS) caused by the activating PIK3CA mutations indicated. (A) Immunoblots, with the truncated p85α Δexon11 variant arrowed; (B)-(E) quantification of immunoblot bands from 3 independent experiments shown for phosphoAKT-S473, phosphoAKT-T308, p110δ and p110α respectively. Paired datapoints +/- insulin are shown in (B) and (C), and dotted lines mark means. Asterisks indicate a significant difference. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure EV2.

Blunted insulin signaling in 3T3-L1 preadipocyte models of APDS2 and SHORT syndrome.

Immunoblotting of Akt, Akt phosphorylated at threonine 308 (T308) or serine 473 (S473), p85α, and p110α and are shown with and without stimulation with 100 nM insulin (Ins) for 10 minutes. Cells were treated with doxycycline (Dox) 1μg/mL for 72 hours prior to insulin stimulation as indicated. (A) One immunoblot representing 3 experiments is shown. (B)-(C) Quantification of immunoblot bands from all 3 independent experiments shown for phosphoAkt-S473 and phosphoAkt-T308 respectively. Paired datapoints +/- insulin are shown, and dotted lines mark means. Asterisks indicate a significant difference. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure EV3. (D) Staining for neutral lipid with Oil Red O of 3T3-L1 cells at day 10 of adipocyte differentiation. Induction of transgene expression by 1μg/mL doxycycline throughout differentiation is shown. Images of entire plates are shown above, with representative bright field microscopy images below.

SHORT syndrome p85α mutations impair phosphotyrosine-stimulated phosphoinositide 3-kinase activity. Lipid kinase activity of purified recombinant PI3K complexes generated using baculoviral expression in Sf9 cells was measured using a modified fluorescence polarisation assay. WT p85α or p85α SHORT syndrome mutations, E489K, R649W or Y657X bound to either (A) p110α (B) p110β or (C) p110δ were assayed for basal and bisphosphotyrosine (pY2)-stimulated lipid kinase activity. Dotted lines mark means, and asterisks indicate a significant difference between the bisphosphotyrosine (pY2)-stimulated state for WT and comparator mutant p85α. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure EV5.

Ability of pathogenic p85α variants to bind p110α, assessed by co- immunoprecipitation. Results of immunoblotting of anti-p110α immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. (B) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments. Co-immunoprecipitated p85α is shown normalized to immunoprecipitated p110α from all 3 independent experiments. Datapoints from the same experiment +/- doxycline are connected by lines. No significant differences were found among conditions.

Attenuated insulin-induced association of p110α with Irs1 in the presence of APDS2 and SHORT syndrome mutant p85α. Results of immunoblotting of anti-Irs1 immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. Treatment with 100nM insulin (Ins) is indicated. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. Two separate sets of gels, including independent wild-type controls are shown on left and right. (B-C) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments. Immunoprecipitated p110α is shown normalized to immunoprecipitated Irs1 from all 3 independent experiments in (B), and immunoprecipitated p85α similarly in (C). Datapoints from the same experiment +/- insulin are connected by lines. Asterisks indicate significant differences induced by transgene overexpression (i.e. plus versus minus doxycycline). More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure EV6.

Attenuated insulin-induced association of p110α with Irs2 in the presence of APDS2 and SHORT syndrome mutant p85α. Results of immunoblotting of anti-Irs2 immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. Treatment with 100nM insulin (Ins) is indicated. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. Two separate sets of gels, including independent wild-type controls are shown on left and right. (B-C) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments. Immunoprecipitated p110a is shown normalized to immunoprecipitated Irs2 from all 3 independent experiments in (B), and immunoprecipitated p85a similarly in (C). Datapoints from the same experiment +/- insulin are connected by lines.

Antibodies Used

Further characterisation of primary dermal fibroblasts studied. (A) Details of cDNA sequence for PIK3CA and PIK3R1 from cells derived from healthy controls (WT), patients with APDS2 (p85a DEx11) or PIK3CA-related overgrowth syndrome (PROS), confirming expected expression of mutant alleles. (B) Higher magnification detail of immunoblot from wild-type and APDS2 fibroblasts showing truncated p85a Dex11. (C) All 3 immunoblot replicates for p110d blots quantified in Figure 1, showing severely reduced p110d expression in the APDS2 cell line

Full statistical analysis of data presented in main

Figure 1 Analysis of insulin-induced increase in (A) AKT S473/4 and (B) T308/9 phosphorylation. The paired mean difference for 3-4 comparisons are shown in Cumming estimation plots. The raw data, as presented in Figure 1, are re-plotted on the upper axes with paired observations connected by a line. On the lower axes, paired mean differences are plotted as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical error bars. (C-D) Analysis of differences in (C) p110a and (D) p110d protein expression between healthy control cells and cells from PROS patients harbouring activating PIK3CA mutations. Mean differences are shown in Gardner-Altman estimation plots, with expression data plotted on the left axes and mean difference on floating axes on the right, again as a bootstrap sampling distribution with mean difference depicted as a dot and 95% confidence intervals by the ends of the vertical bar.

Full statistical analysis of data presented in main

Figure 2. Analysis of the effects of doxycycline-induced expression of wild-type (WT) or ΔEx11 (APDS2) p85α, or of p110α H1047R (PROS) on Akt S473/4 (A,B) and T308/9 (C,D) phosphorylation. Comparisons are made in both the basal, non-insulin stimulated state (A,C) and after stimulation with 10 nmol/L insulin (B,D). Paired mean differences for 3 comparisons are shown in Cumming estimation plots. Raw data, as presented in Figure 2, are re-plotted on the upper axes with paired observations connected by a line. On the lower axes, paired mean differences are plotted as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical error bars.

The effect of graded expression of wild-type or disease- associated p85α on 3T3-L1 preadipocytes. Immunoblots of p85α, phosphoAkt (S473) and total Akt are shown for control 3T3-L1 cells and 3T3-L1 cells conditionally expressing wild- type (WT), or APDS2-associated mutant p85α under the control of doxycycline (Dox), with and without 10 minutes of exposure to insulin as indicated. The filled black triangles indicate increasing concentrations of doxycycline (from left to right: 0, 0.02, 0.03, 0.045, 0.065, or 0.1 μg/mL). Exposure was for 72 hours in all cases. The truncated p85α variant can be seen below the WT p85α for the APDS2 ΔEx11 mutant.

Full statistical analysis of data presented in main

Figure 3. Analysis of fluorescence polarisation assay of phosphoinositide 3-kinase (PI3K) activity of in vitro synthesised wild-type (WT) or mutant (E489K, R649W or Y657X) p85α. Results for p110α- and p110β-containing PI3K are shown in (A) and (B) respectively. All data were acquired in the presence of phosphotyrosine peptide. Paired mean differences for 3 comparisons are shown in Cumming estimation plots. Raw data, as presented in Figure 3, are re-plotted on the upper axes with paired observations connected by a line. On the lower axes, paired mean differences are plotted as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical error bars. Results for the R649W p85α mutation only are shown with p110δ in (C). In this case raw data are re-plotted on the left hand axes with paired observations connected by 3 nearly superimposed lines. On the right hand axes, paired mean differences are plotted as a bootstrap sampling distribution.

Full statistical analysis of data presented in main

Figure 5. Analysis of the effects of doxycycline (dox)-induced expression of wild-type (WT), Y657X, R649W or ΔEx11 p85α on association of p110α (A,B) and p85α (C,D) with Irs1. Results of co-immunoprecipitation with (B,D) and without exposure to 10 nmol/L insulin Comparisons are made in both the basal, non-insulin stimulated state (A,C) and after stimulation with 10 nmol/L insulin (B,D) are shown. Paired mean differences for 3 comparisons are shown in Cumming estimation plots. Raw data, as presented in Figure 5, are re-plotted on the upper axes with paired observations connected by a line. On the lower axes, paired mean differences are plotted as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical error bars.

Full statistical analysis of data presented in Figure EV7.

Analysis of the effects of doxycycline (dox)-induced expression of wild-type (WT), Y657X, R649W or ΔEx11 p85α on association of p110α (A,B) and p85α (C,D) with Irs2. Results of co-immunoprecipitation with (B,D) and without exposure to 10 nmol/L insulin Comparisons are made in both the basal, non-insulin stimulated state (A,C) and after stimulation with 10 nmol/L insulin (B,D). Paired mean differences for 3 comparisons are shown in Cumming estimation plots. Raw data, as presented in Figure EV7, are re-plotted on the upper axes with paired observations connected by a line. On the lower axes, paired mean differences are plotted as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical error bars.