Journal: bioRxiv

Article Title: Insulin receptor substrate 2 (IRS2) confers resistance to PI3K pathway inhibition in PIK3CA mutant breast cancer

doi: 10.64898/2026.04.24.720709

Figure Lengend Snippet: (A and B) PIK3CA WT and PIK3CA mutant knockin MCF10A cells were serum starved, then evaluated for IRS1 and IRS2 mRNA (A) and protein (B) expression. The data shown in the graphs represent the mean +/- standard deviation (SD) of three independent experiments. Statistical analysis by One-Way ANOVA followed by Dunnett’s multiple comparisons test to compare mutants against WT (P values shown). (C and D) PIK3CA WT and PIK3CA mutant knockin MCF10A cells were treated in plain media with or without alpelisib (5μM) or MK2206 (1μM) for 24 hrs and then evaluated for IRS1 or IRS2 mRNA (C) or protein (D) expression. The data shown in the graphs represent the mean +/- SD of three independent experiments. Statistical analysis by (C) separate One-Way ANOVA followed by the Bonferroni test for multiple comparisons to compare drug treatments to DMSO or (D) Two-Way ANOVA followed by the Tukey test for multiple comparisons to compare all treatments within each cell type (P values shown). (E) IRS1 and IRS2 protein expression in PIK3CA WT and PIK3CA mutant breast epithelial and cancer cell lines following serum starvation (M10A - MCF10A, U812 - UACC812, MDA-231 – MDA-MB-231, H1806 - HCC1806, SUM159 - SUM159PT). (F) PIK3CA mutant breast cancer cells were treated in plain media with or without alpelisib (5μM) for 24 hrs and cell extracts were evaluated by immunoblot. (G and H) Differential IRS1 and IRS2 mRNA (G) and protein (H) in breast cancer cell lines from DepMap. (mRNA - log2(TPM+1), protein RPPA signal). The data shown in the graphs represent the mean +/- SD of all included cell lines. Percent changes were determined by comparing the means of the original data (prior to log transformation). Statistical analysis by Welch’s t test (two-tailed) (P values shown). See also Figure S2.

Article Snippet: MK2206 was prepared at 25mM in DMSO prior to diluting for experiments (MedChemExpress).

Techniques: Mutagenesis, Knock-In, Expressing, Standard Deviation, Western Blot, Transformation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Insulin receptor substrate 2 (IRS2) confers resistance to PI3K pathway inhibition in PIK3CA mutant breast cancer

doi: 10.64898/2026.04.24.720709

Figure Lengend Snippet: (A) MDA-MB-231 cells and (B) SUM159PT cells were treated with non-targeting guide RNA ( NT ) or IRS2-targeting guide RNA (IRS2KO) and cell extracts were analyzed by immunoblot. (C) Viability (live cell population size) of PIK3CA WT MDA-MB-231 NT and IRS2KO cells treated with increasing concentrations of alpelisib or MK2206 for 72 hrs, measured by crystal violet staining. (D) Viability of PIK3CA mutant SUM159PT NT and IRS2KO cells treated with increasing concentrations of alpelisib or MK2206 for 72 hrs, measured by crystal violet staining. (C and D) Statistical analysis by variable slope (four parameters) nonlinear regression using least squares regression to fit the curve (with asymmetrical confidence intervals calculated for parameters) and the extra sum-of-squares F test to compare IC50 values. Data shown are representative of three independent experiments. See also Figure S4.

Article Snippet: MK2206 was prepared at 25mM in DMSO prior to diluting for experiments (MedChemExpress).

Techniques: Western Blot, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Insulin receptor substrate 2 (IRS2) confers resistance to PI3K pathway inhibition in PIK3CA mutant breast cancer

doi: 10.64898/2026.04.24.720709

Figure Lengend Snippet: (A,C,E) Relative Viability (top graph) and Lethal Fraction (bottom graph) of NT and IRS2KO PIK3CA WT (A), PIK3CA E545K (C), and PIK3CA H1047R (E) MCF10A cells treated with increasing concentrations of alpelisib for 72 hrs, measured by FLICK assay. (B,D,F) Relative Viability (top) and Lethal Fraction (bottom) of NT and IRS2KO PIK3CA WT (B), PIK3CA E545K (D), and PIK3CA H1047R (F) MCF10A cells treated with increasing concentrations of MK2206 for 72 hrs, measured by FLICK assay. Statistical analysis by variable slope (four parameters) nonlinear regression using least squares regression to fit the curve (with asymmetrical confidence intervals calculated for parameters) and the extra sum-of-squares F test to compare IC50 values for all panels except Lethal Fraction for (A) and (B). Statistical analysis for Lethal Fraction for (A) and (B) by measuring area under curve (AUC) (GraphPad Prism). AUCs were compared using Welch’s t test (two-tailed), utilizing AUC total area and standard error. For AUC comparison of Lethal Fraction for (A) and (B), P values shown are for comparison of data after normalization to lowest drug dose for each subline. P values for original data (shown in graphs) are p<0.0001 (A and B). For all panels, data shown are representative of three independent experiments. See also Figure S5.

Article Snippet: MK2206 was prepared at 25mM in DMSO prior to diluting for experiments (MedChemExpress).

Techniques: Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Insulin receptor substrate 2 (IRS2) confers resistance to PI3K pathway inhibition in PIK3CA mutant breast cancer

doi: 10.64898/2026.04.24.720709

Figure Lengend Snippet: (A and B) Relative Viability (top graph) and Lethal Fraction (bottom graph) of NT and IRS2KO PIK3CA H1047L mutant SUM159PT cells treated with increasing concentrations of alpelisib (A) or MK2206 (B) for 72 hrs, measured by FLICK assay. Statistical analysis by variable slope (four parameters) nonlinear regression using least squares regression to fit the curve (with asymmetrical confidence intervals calculated for parameters) and the extra sum-of-squares F test to compare IC50 values for Relative Viability (A and B). Statistical analysis for Lethal Fraction for (A) and (B) by measuring area under curve (AUC) (GraphPad Prism). AUCs were compared using Welch’s t test (two-tailed), utilizing AUC total area and standard error. Data shown are representative of three independent experiments. (C) NT and IRS2KO PIK3CA H1047L mutant SUM159PT cells were treated with increasing concentrations of alpelisib for 24 hrs and cell extracts were evaluated by immunoblot. The data shown in the graphs represent the mean +/- SD of three independent experiments. Statistical analysis was performed using a natural-log transformation followed by ordinary least squares (OLS) ANOVA on log(ratio), with fixed effects for genotype (NT vs IRS2KO), dose (categorical: 0 (DMSO), 0.1, 0.5, 1, 2.5, 10), their interaction, and a gel blocking factor, according to the model: log(ratio) ∼ genotype × dose + gel. Pre-specified comparisons between NT and IRS2KO were conducted within each dose on the log scale. Because these comparisons were planned a priori, no multiplicity adjustments were applied (P values shown).

Article Snippet: MK2206 was prepared at 25mM in DMSO prior to diluting for experiments (MedChemExpress).

Techniques: Mutagenesis, Two Tailed Test, Western Blot, Transformation Assay, Blocking Assay

Journal: Science Advances

Article Title: Adissp activates insulin-independent glucose disposal and energy expenditure in white fat to treat diabetes and cardiometabolic disease

doi: 10.1126/sciadv.aed2780

Figure Lengend Snippet: ( A ) Phosphorylation of PDK1, Akt, and AS160 from 3-month-old male lean mice after rAdissp (70 nmol/kg, 3 hours) or insulin (0.75 U/kg, 45 min) ( n = 4 per group). ( B ) Blood glucose in 2-month-old male lean mice treated with rAdissp (70 nmol/kg) for 3 hours ( n = 4 per group). ( C and D ) Circulating insulin (C) and glucagon (D) from mice in (B) ( n = 4 per group). ( E ) After a 6-hour fast, male lean mice received an oral glucose load (2 g/kg) plus intraperitoneal rAdissp (50 nmol/kg). Insulin was measured. n = 5 mice per group. ( F ) Ad libitum-fed male lean mice received intraperitoneally insulin (0.5 U/kg) plus rAdissp (50 nmol/kg). Glucagon was measured. n = 5 mice per group. ( G ) Human rAdissp (60 nmol/kg per day) was administrated to ob/ob mice, and blood glucose was measured. rAdissp, n = 14 (7 male and 7 female mice); vehicle, n = 15 (7 male and 8 female mice). ( H ) Circulating insulin ( n = 7 per group) from male ob/ob mice. ( I ) Blood glucose measured 6 hours after rAdissp injection in 7-week-old BTBR ob/ob male mice. n = 5 to 6 per group. ( J ) Representative polyuria images. ( K ) Twelve-hour fasting, 2-month-old male lean mice received rAdissp (80 nmol/kg) and fasting glucose was measured. n = 4 per group. ( L ) Blood glucose in male DIO mice before and 3 hours after rAdissp (60 nmol/kg) injection following a 3-day pretreatment with MK2206 (50 mg/kg). Vehicle, n = 5; MK2206 + Vehicle, n = 5, Vehicle + rAdissp, n = 5; MK2206 + rAdissp, n = 8. ( M and N ) 2-DG uptake in indicated tissues from lean (M; rAdissp 80 nmol/kg, 3 hours) and ob/ob mice [(N), 45 nmol/kg, 3 hours]. n = 3 per group. Two-tailed Student’s t test for [(B), (H), (K), (M), and (N)] and two-way ANOVA for [(G), (I), and (L)].

Article Snippet: To assess whether the glucose-lowering effect of rAdissp is Akt-dependent, the Akt inhibitor MK2206 (MedChemExpress, catalog no. HY-10358) was administered to DIO mice via intraperitoneal injection at 50 mg/kg daily for three consecutive days, followed by a single dose of rAdissp (60 nmol/kg).

Techniques: Phospho-proteomics, Injection, Two Tailed Test

Journal: Science Advances

Article Title: Adissp activates insulin-independent glucose disposal and energy expenditure in white fat to treat diabetes and cardiometabolic disease

doi: 10.1126/sciadv.aed2780

Figure Lengend Snippet: ( A ) Phosphorylation of PDK1, Akt, and AS160 from 3-month-old male lean mice after rAdissp (70 nmol/kg, 3 hours) or insulin (0.75 U/kg, 45 min) ( n = 4 per group). ( B ) Blood glucose in 2-month-old male lean mice treated with rAdissp (70 nmol/kg) for 3 hours ( n = 4 per group). ( C and D ) Circulating insulin (C) and glucagon (D) from mice in (B) ( n = 4 per group). ( E ) After a 6-hour fast, male lean mice received an oral glucose load (2 g/kg) plus intraperitoneal rAdissp (50 nmol/kg). Insulin was measured. n = 5 mice per group. ( F ) Ad libitum-fed male lean mice received intraperitoneally insulin (0.5 U/kg) plus rAdissp (50 nmol/kg). Glucagon was measured. n = 5 mice per group. ( G ) Human rAdissp (60 nmol/kg per day) was administrated to ob/ob mice, and blood glucose was measured. rAdissp, n = 14 (7 male and 7 female mice); vehicle, n = 15 (7 male and 8 female mice). ( H ) Circulating insulin ( n = 7 per group) from male ob/ob mice. ( I ) Blood glucose measured 6 hours after rAdissp injection in 7-week-old BTBR ob/ob male mice. n = 5 to 6 per group. ( J ) Representative polyuria images. ( K ) Twelve-hour fasting, 2-month-old male lean mice received rAdissp (80 nmol/kg) and fasting glucose was measured. n = 4 per group. ( L ) Blood glucose in male DIO mice before and 3 hours after rAdissp (60 nmol/kg) injection following a 3-day pretreatment with MK2206 (50 mg/kg). Vehicle, n = 5; MK2206 + Vehicle, n = 5, Vehicle + rAdissp, n = 5; MK2206 + rAdissp, n = 8. ( M and N ) 2-DG uptake in indicated tissues from lean (M; rAdissp 80 nmol/kg, 3 hours) and ob/ob mice [(N), 45 nmol/kg, 3 hours]. n = 3 per group. Two-tailed Student’s t test for [(B), (H), (K), (M), and (N)] and two-way ANOVA for [(G), (I), and (L)].

Article Snippet: To assess whether the glucose-lowering effect of rAdissp is Akt-dependent, the Akt inhibitor MK2206 (MedChemExpress, catalog no. HY-10358) was administered to DIO mice via intraperitoneal injection at 50 mg/kg daily for three consecutive days, followed by a single dose of rAdissp (60 nmol/kg).

Techniques: Phospho-proteomics, Injection, Two Tailed Test

Journal: Science Advances

Article Title: Adissp activates insulin-independent glucose disposal and energy expenditure in white fat to treat diabetes and cardiometabolic disease

doi: 10.1126/sciadv.aed2780

Figure Lengend Snippet: ( A to E ) Male DIO mice received daily rAdissp (68 nmol/kg) for 33 days. Blood glucose was measured 24 hours after each injection under ad libitum conditions (day 1 included a 2-hour fast) ( n = 19 to 20 per group) (A). Daily food intake (B) and circulating insulin (C) were assessed. Insulin tolerance tests (ITTs; insulin, 0.75 U/kg) (D) and glucose tolerance tests (GTTs; glucose, 0.1 g per mouse) (E) were performed. ( F to J ) Twelve-week-old ob/ob mice were treated daily with rAdissp (60 nmol/kg) for 30 days. Blood glucose was measured 24 hours after each injection. Vehicle group, n = 14 (6 male and 8 female mice); treated group, n = 16 (8 male and 8 female mice) (F). Daily food intake was recorded ( G ). Circulating insulin was measured in male mice ( n = 6 to 8 per group) and female mice ( n = 8 per group) ( H ). ITT (insulin, 0.75 U/kg) ( I ) and GTT (glucose, 0.06 g per mouse) ( J ) were conducted (vehicle group, n = 14; treated group, n = 16). ( K to N ) Following high-dose STZ, male mice received a single rAdissp dose, and glucose was measured over time after an 8- or 6-hour fast ( n = 9 to 10 per group) (K), with representative polyuria images (L). After low-dose STZ, male mice were treated daily with rAdissp (72 nmol/kg) for 13 days; glucose was measured 24 hours postinjection (day 1 included a 2-hour fast) ( n = 8 per group) (M), with representative polyuria images (N). Two-tailed Student’s t test for [(C) and (H)] and a two-way ANOVA for [(A), (D), (E), (F), (I), (J), (K), and (M)].

Article Snippet: To assess whether the glucose-lowering effect of rAdissp is Akt-dependent, the Akt inhibitor MK2206 (MedChemExpress, catalog no. HY-10358) was administered to DIO mice via intraperitoneal injection at 50 mg/kg daily for three consecutive days, followed by a single dose of rAdissp (60 nmol/kg).

Techniques: Injection, Two Tailed Test

Journal: Science Advances

Article Title: Adissp activates insulin-independent glucose disposal and energy expenditure in white fat to treat diabetes and cardiometabolic disease

doi: 10.1126/sciadv.aed2780

Figure Lengend Snippet: ( A ) Representative images of body surface temperature in male DIO mice after five weeks of daily rAdissp (68 nmol/kg) treatment. ( B ) Core body temperature in the mice from (A) ( n = 19 to 20 per group). ( C and D ) Body weight gain and EchoMRI in the mice from (A) ( n = 19 to 20 per group). ( E and F ) Representative liver images (E) and hematoxylin and eosin (H&E) staining (F) from the mice in (A). Scale bar, 200 μm. ( G and H ) Liver triglyceride content [(G), n = 10 per group] and weight [(H), n = 10 to 19] from mice in (A). ( I ) Serum levels of AST and ALT from mice in (A) ( n = 19 to 20 per group). ( J to M ) Heart triglyceride levels [(J), n = 10 per group], weight [(K), n = 19 to 20], and representative images [(L) and (M)] from the mice in (A). ( N and O ) Serum triglyceride ( n = 10 per group) and uric acid levels ( n = 19 to 20 per group) from the mice in (A). ( P to Z ) ob/ob mice received daily rAdissp (60 nmol/kg) for 1 month. Representative images of body surface temperature (P). Body weight gain [(Q), vehicle: n = 14, 6 male and 8 female mice; rAdissp: n = 16, 8 male and 8 female mice]. In male ob/ob mice, liver images (R), H&E staining [(S), scale bars = 200 μm], triglycerides [(T), n = 6 to 8 per group], weight [(U), n = 6 to 8 per group], and serum AST/ALT [(V), n = 6 to 8 per group] were determined. Heart triglycerides [(W), n = 6 to 8 per group], weight [(X), n = 6 to 8 per group], H&E staining (Y), and serum triglycerides [(Z), n = 6 to 8 per group] were measured. Two-tailed Student’s t test was performed, except for (C), where a two-way ANOVA was performed. Scale bar, 200 μm.

Article Snippet: To assess whether the glucose-lowering effect of rAdissp is Akt-dependent, the Akt inhibitor MK2206 (MedChemExpress, catalog no. HY-10358) was administered to DIO mice via intraperitoneal injection at 50 mg/kg daily for three consecutive days, followed by a single dose of rAdissp (60 nmol/kg).

Techniques: Staining, Two Tailed Test