Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Effect of prolonged treatment with afatinib on expression of membrane transport proteins involved in chloride secretion. ( a ) Effect on mRNA expression. qRT-PCR was performed to measure mRNA expression of TMEM16A, CFTR, potassium calcium-activated channel subfamily N member 4 (K Ca 3.1), potassium voltage-gated channel subfamily Q member 1 (K v 7.1), and sodium potassium chloride cotransporter 1 (NKCC1), normalized by vehicle, after treatment of AFT (0.1 µM) or vehicle for 24 h ( n = 3–4). Western blot analyses were performed to investigate the protein expression of NKCC1 ( b ), K v 7.1( c ), and TMEM16A ( d ) after treatment with AFT or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S3. Results are means ± S.E.M. ( n = 3 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. All results were analyzed by Student t-test.

Article Snippet: Following membrane staining, colonoids were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Then, they were incubated overnight at 4 °C with primary antibodies targeting TMEM16A (1:50 dilution, bs3794R, Bioss Inc., Woburn, MA, USA).

Techniques: Expressing, Membrane, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Role of PI3K in afatinib-induced alteration of transport protein expression and secretory lineage differentiation. ( a ) Confirmation of PI3K inhibition by afatinib. Western blot analysis was performed to measure the protein expression of P-PI3K in colonoids treated with AFT (0.1 µM) or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S4. Results are means ± S.E.M. ( n = 3). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by Student t-test. ( b ) Role of PI3K in mediating the effect of afatinib on mRNA expression. Colonoids were treated with vehicle, AFT, BAY-80-6946 (50 nM; a PI3K inhibitor) or AFT plus BAY-80-6946 for 24 h before mRNA extraction and qRT-PCR being performed to analyze mRNA expression of NKCC1, K v 7.1, TMEM16A, ATOH1, and LYZ1. Results are means ± S.E.M. ( n = 3–7 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by ANOVA followed by Turkey’s post hoc test.

Article Snippet: Following membrane staining, colonoids were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Then, they were incubated overnight at 4 °C with primary antibodies targeting TMEM16A (1:50 dilution, bs3794R, Bioss Inc., Woburn, MA, USA).

Techniques: Expressing, Inhibition, Western Blot, Extraction, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Effect of prolonged treatment with afatinib on expression of membrane transport proteins involved in chloride secretion. ( a ) Effect on mRNA expression. qRT-PCR was performed to measure mRNA expression of TMEM16A, CFTR, potassium calcium-activated channel subfamily N member 4 (K Ca 3.1), potassium voltage-gated channel subfamily Q member 1 (K v 7.1), and sodium potassium chloride cotransporter 1 (NKCC1), normalized by vehicle, after treatment of AFT (0.1 µM) or vehicle for 24 h ( n = 3–4). Western blot analyses were performed to investigate the protein expression of NKCC1 ( b ), K v 7.1( c ), and TMEM16A ( d ) after treatment with AFT or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S3. Results are means ± S.E.M. ( n = 3 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. All results were analyzed by Student t-test.

Article Snippet: The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, washed with Tris-buffered saline with 1% Tween (TBST) and incubated overnight at 4 °C with antibodies to NKCC1 (14581), K v 7.1 (N37A/10, Invitrogen, Waltham, MA, USA), p-PI3K (17366 S), TMEM16A (bs3794R, Bioss Inc., Woburn, MA, USA) or β-actin (4970) at 1:1000 dilution.

Techniques: Expressing, Membrane, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Role of PI3K in afatinib-induced alteration of transport protein expression and secretory lineage differentiation. ( a ) Confirmation of PI3K inhibition by afatinib. Western blot analysis was performed to measure the protein expression of P-PI3K in colonoids treated with AFT (0.1 µM) or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S4. Results are means ± S.E.M. ( n = 3). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by Student t-test. ( b ) Role of PI3K in mediating the effect of afatinib on mRNA expression. Colonoids were treated with vehicle, AFT, BAY-80-6946 (50 nM; a PI3K inhibitor) or AFT plus BAY-80-6946 for 24 h before mRNA extraction and qRT-PCR being performed to analyze mRNA expression of NKCC1, K v 7.1, TMEM16A, ATOH1, and LYZ1. Results are means ± S.E.M. ( n = 3–7 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by ANOVA followed by Turkey’s post hoc test.

Article Snippet: The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, washed with Tris-buffered saline with 1% Tween (TBST) and incubated overnight at 4 °C with antibodies to NKCC1 (14581), K v 7.1 (N37A/10, Invitrogen, Waltham, MA, USA), p-PI3K (17366 S), TMEM16A (bs3794R, Bioss Inc., Woburn, MA, USA) or β-actin (4970) at 1:1000 dilution.

Techniques: Expressing, Inhibition, Western Blot, Extraction, Quantitative RT-PCR