Figure 1. KChIP3 levels regulate baseline MUC5AC secretion.

(A) KChIP3 RNA levels from undifferentiated (UD) and differentiated (DF) HT29-18N2 cells normalized by GAPDH values. (B) Control (black circles) and KChIP3 stable knockdown cells (KChIP3-KD) (blue squares) were differentiated and incubated for 30 min at 37°C in the absence or presence of 100 µM ATP. Secreted MUC5AC was collected and dot blotted with an anti-MUC5AC antibody. Data were normalized to actin levels. The y-axis represents normalized values relative to the values of untreated control cells. (C) ATP-dependent MUC5AC secretion was calculated from the data in (B) as the difference between normalized baseline secretion and stimulated secretion for each condition. (D) Secreted MUC5AC from differentiated control (black circles) and KChIP3 overexpressing cells (KChIP3-GFP) (red circles) in the absence or presence of 100 µM ATP. (E) ATP-dependent MUC5AC secretion calculated from the data in (D) for each condition. (F) Immunofluorescence Z-stack projections of control, KChIP3-KD and KChIP3-GFP differentiated HT29-18N2 cells with anti-MUC5AC antibody (green) and DAPI (red). Scale bar = 5 µm. (G) The number of MUC5AC granules for control (black circles), KChIP3-KD (blue squares) and KChIP3-GFP (red circles) cells was quantified from individual immunofluorescence stacks using 3D analysis FIJI software. The y-axis represents the number of 3-D objects detected by the software divided by the number of cells in each field. (H) Volume of control (black), KChIP3-KD (blue) and KChIP3-GFP (red) MUC5AC granules was calculated from individual immunofluorescence stacks using 3D analysis FIJI software. The y-axis represents the volume of the granules in µm³. Abbreviations: UD: Undifferentiated HT29-18N2 cells, DF: Differentiated HT29-18N2 cells. *p<0.05, **p<0.01.

Figure 1—figure supplement 1. KChIP expression levels in HT29-18N2 stable cell lines.

(A) KCNIP1 (KChIP1), KCNIP2 (KChIP2), KCNIP3 (KChIP3) and KCNIP4 (KChIP4) RNA levels normalized to values of GAPDH from control and KChIP3-KD cells. mRNA levels of each gene are represented as relative value compared to control cells. Results are average values ± SEM (N ≥ 3). (B) Cell lysates from control, KChIP3-GFP and KChIP3-MUT HT29-18N2 differentiated cells were analysed by western blot with an anti-KChIP3 and an anti-GFP antibody to test expression levels. Actin was used as a loading control. (C) RNA levels of KCNIP1 (KChIP1), KCNIP2 (KChIP2), KCNIP3 (KChIP3) and KCNIP4 (KChIP4) (normalized to values of the GAPDH) from control and KChIP3-GFP cells. mRNA levels of each gene are represented as relative value compared to control cells. Results are average values ± SEM (N ≥ 3). Abbreviations: CTRL: control cells. **p<0.01.

Figure 1—figure supplement 2. KChIP3 levels regulate MUC2 secretion.

(A) Secreted MUC2 from differentiated control (black circles) and KChIP3 stable knockdown cells (KChIP3-KD) (blue squares) after 30 min incubation at 37°C in normal buffer (1.2 mM CaCl₂). Data were normalized to actin levels. The y-axis represents normalized values relative to the values of untreated control cells. Average values +- SEM are plotted as scatter plot with bar graph (N = 3). (B) Secreted MUC2 from differentiated control (black circles), KChIP3-GFP overexpressing cells (KChIP3-GFP) (red circles) and KChIP3-MUT overexpressing cells (KChIP3-MUT) (green circles) after 30 min incubation at 37°C in normal buffer (1.2 mM CaCl₂). Data were normalized to actin levels. The y-axis represents normalized values relative to the values of untreated control cells. Average values ± SEM are plotted as scatter plot with bar graph (N = 3). (C) MUC5AC internal protein levels in differentiated control and KChIP3-KD cells normalized to the actin levels. Average values ± SEM are plotted as scatter plot with bar graph (N = 3). (D) MUC5AC internal levels in differentiated control, KChIP3-GFP and KChIP3-MUT cells normalized to actin levels. Average values ± SEM are plotted as scatter plot with bar graph (N = 3). *p<0.05, **p<0.01.