Fig 2: Imbalance of d-rap1 activity leads to loss of slit diaphragms and lacunae. (A) TEM analysis of dissected control nephrocytes (sns>>control OE), nephrocytes with overexpression of Drosophila wild-type rap1 (sns>>d-rap1 WT), of Drosophila dominant-negative rap1 (sns>>d-rap1 N17), or of Drosophila constitutively active rap1 (sns>>d-rap1 V12). Knockdown in nephrocytes was accomplished by employing sns-GAL4. White arrows exemplarily indicate slit diaphragms. Scale bars: upper panel 500 nm, lower panel zoom compared to the upper panel 2×. n = 3. (B) Statistical evaluation of the number of slit diaphragms (SD) per µm basement membrane of the genotypes shown in (A) depicted as a box plot with minimum and maximum. The control is marked in pink. **p < 0.01, ***p < 0.001.

Fig 3: Rap1 functions downstream of nephrin in signaling to integrin β. Immunofluorescence analysis of tangential (A) or surface sections (B) of nephrocytes overexpressing sns (sns>>UAS Sns x control RNAi) or nephrocytes with overexpression of sns and knockdown of rap1 (sns>>UAS Sns x Rap1 RNAi 20761 or sns>>UAS Sns x Rap1 RNAi 110757 ). Knockdown in nephrocytes was accomplished by employing sns-GAL4. Wandering third instar larvae were dissected, and immunofluorescence analysis was performed with antibodies specific for integrin β (green) and Sns (red). Merged images and higher magnifications of the marked area are shown. Scale bars (A,B): 10 µm, n = 3. (C) Statistical evaluation of the number of slit diaphragms (SD) per µm nephrocyte surface area of the genotypes shown in (A,B) depicted as a box plot with minimum and maximum. The control is marked in pink. ***p < 0.001.

Fig 4: Rap1b/talin-F0 interaction is GTP-dependent and specific but is of weak affinity. a The HSQC spectra of 50 μM 15N-labeled talin-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red). b The HSQC spectra (four representative residues were shown) of 50 μM 15N-labeled talin-F0 in the absence (black) and presence of 125 μM GDP loaded Rap1b (blue) or GMP-PNP loaded Rap1b (red). Note that Rap1b-GDP induced the same overall pattern of chemical shift changes of 15N-labeled talin-F0 as Rap1b-GMP-PNP does but with less peak shifts and broadenings, indicating a weaker affinity. c The affinity of Rap1b (GMP-PNP)/talin-F0 interaction measured by HSQC titration. d The HSQC spectra of 50 μM 15N-labeled kindlin2-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red)

Fig 5: Rap1/PI3K-Akt signaling pathway correlates with Gemcitabine-resistance in PDAC cells. (A) Schematic of the differentially expressed gene discovery in Gem-R PDAC cells using GSE148200 and GSE140077. (B) Venn-diagram of up- and down-regulated expression (log2FC > ±1.0 and p < 0.01) of the genes in Gem-R PDAC cells. (C,D) Scatter plot of KEGG pathway enrichment analysis of up- (C) and down-regulated (D) genes in Gem-R PDAC cells. The number of differentially expressed genes in the pathway is indicated by the circle area, and the circle color represents the range of p value. (E) qRT-PCR analysis of key differentially expressed genes of Rap1/PI3K-Akt signaling pathway in parental and Gem-R PDAC cells. β-Actin mRNA expression was used as an internal control. The average (column) ± SD is indicated (* p < 0.05 vs. parental). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; FC, fold change; SD, standard deviation.