Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Chlamydomonas expressing KAP-GFP. Indicated are the measured areas of KAP-GFP localization: the two basal bodies and cilia. Scale bar is 5 μm. (B) Super plot quantification of basal body fluorescence in (A) after a 2-h treatment with BCI at the indicated concentrations. Error bars are the mean with the 95% confidence interval for the averages from the trials. P = 0.5126, which was determined by an ordinary one-way ANOVA. (C) Ciliary length plotted against ciliary fluorescence for cells in DMSO (orange circles) or 30 μM BCI (purple) after a 2-h treatment (n = 40, N = 1). Simple linear regression was used to generate linear regression lines and comparisons. For DMSO, y = 0.1171x + 0.4305 and r 2 = 0.05676. For BCI, y = 0.1174x + 0.4051 and r 2 = 0.2636. Comparing slopes, F = 4.048e-006 (1, 76) and P = 0.9984. Comparing intercepts, F = 0.005429 (1, 77) and P = 0.9415. (D) Comparison of total ciliary fluorescence shown in (C). P -values were determined using a t test. (E) Comparisons of ciliary fluorescence in DMSO or BCI per micrometer of cilia in (C). P -values were determined using a t test. (F) Example kymographs collected from total internal reflection fluorescent microscopy of KAP-GFP movement in cilia in cells treated with DMSO (top) or 30 μM BCI (bottom) for 2 h. Vertical scale bars are 4 μm. Horizontal scale bars are 2 s. (G, H, I) KAP-GFP dynamics quantified from the kymographs (n = 20, N = 1). Error bars are the mean with the 95% confidence interval (n = 20, N = 1). P -values were calculated from a two-tailed unpaired t test for pairwise comparisons. (G) Frequency of KAP-GFP trains measured as the total amount of trains counted over the total amount of time the kymograph was collected. For DMSO, n = 40 (1 and 2 h). For BCI, n = 30 (1 h) and 34 (2 h). (H) Velocity of KAP-GFP trains measured as the distance traveled in μm over time in seconds. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). (I) Relative injection size of KAP-GFP trains measured as the relative total fluorescent intensity of each train relative to the maximum measurement. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). P -values were determined using a t test.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Two Tailed Test, Injection

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Immunofluorescent images of KAP-GFP cells during regeneration in either DMSO or 30 μM BCI. Scale bars are 5 μm. Red arrows point to basal bodies, which are the object of quantification in (D). (B) Western blot for KAP-GFP expression in regenerating cells in either DMSO (D) or 30 μM BCI (B). Total protein was measured with amido black. (C) Quantification of (B). Error bars are the mean with SD for three independent experiments. P -values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (D) Quantification of KAP-GFP fluorescence at the basal bodies in (A). Error bars are the mean with the 95% confidence interval for the averages from three independent trials (n = 30, N = 3). The P -value was calculated using an unpaired t test with Welch’s correction.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Western blot of KAP-GFP expression compared with total protein shown with amido black. Cells were treated for 2 h with either 0.5% DMSO, 20 μM BCI, 50 μM MG132, or both 50 μM MG132 and 20 μM BCI. (A, B) Quantification of (A). Error bars are SD of the mean (N = 3). The P -value was determined by an unpaired t test between BCI and BCI + MG132.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: BCI inhibits phosphate removal from MAPK. This ultimately alters or inhibits ciliogenesis, KAP-GFP dynamics in cilia, KAP-GFP protein synthesis, NPHP4 protein localization at the transition zone, membrane trafficking, and microtubule organization.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques:

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: Tools and reagents.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Staining, Mutagenesis