Fig 1: JNK inhibition reduces tubule cell proliferation in juvenile Pkd2 mutant mice.Mice with the following genotypes were treated with tamoxifen by maternal transfer at P2-4 and collected at P21: Con (Rosa26-CreERT2; Pkd2fl/+), Pkd2 (Rosa26-CreERT2; Pkd2fl/fl; Jnk1+/+, fl/+; Jnk2+/+, +/-), and Pkd2-Jnk (Rosa26-CreERT2; Pkd2fl/fl; Jnk1fl/fl; Jnk2null/null). (A) Kidney sections from P21 mice were probed for the mitotic marker phospho S10 histone H3 along with tubule epithelial markers LTA (proximal tubules) and DBA (collecting ducts). Nuclei were marked with DAPI. Images are maximum projection of z-stacks (10 slices at 0.5um intervals) obtained on Zeiss LSM 900 Airyscan microscope with 20X objective. Scale bar is 50 microns and applies to all images in the panel. (B-C) Quantification of the proportion of tubule cells with nuclei positive for phospho S10 histone H3. (B) Proximal tubule cells (LTA+) and (C) collecting duct cells (DBA+) were quantified separately. N is 5 animals per group, 1000–2000 cells per animal. ****, P < 0.0001; ***, P< 0.001; *, P < 0.05; ns, not significant by one-way ANOVA followed by Tukey multiple comparison test with multiplicity-adjusted p-values. Error bars indicate SD.
Fig 2: Pkd2 deletion activates c-Jun in kidney tubule epithelial cells.Mice with the following genotypes were treated with tamoxifen by maternal transfer at P2-4 and collected at P21: Con (Rosa26-CreERT2; Pkd2fl/+), Pkd2 (Rosa26-CreERT2; Pkd2fl/fl; Jnk1+/+, fl/+; Jnk2+/+, +/-), and Pkd2-Jnk (Rosa26-CreERT2; Pkd2fl/fl; Jnk1fl/fl; Jnk2null/null). (A) Kidney sections were probed for phospho S63 c-Jun and tubule epithelial markers LTA (proximal tubules) or DBA (collecting ducts). Examples of non-cystic and cystic collecting ducts are shown. Nuclei were marked by DAPI. Images are maximum projection of z-stacks (20 slices at 0.5um intervals) obtained on Zeiss LSM 900 Airyscan microscope with 40X objective. Scale bar is 20 microns and applies to all images in the panel. (B-C) Quantification of the proportion of tubule epithelial cells with nuclei positive for phospho S63 c-Jun. (B) Proximal tubules (LTA+) and (C) collecting ducts (DBA+) were quantified separately. N is 5 animals per group, 1000–2000 cells per animal. ****, P < 0.0001; ***, P< 0.001; *, P < 0.05; ns, not significant by one-way ANOVA followed by Tukey multiple comparison test with multiplicity-adjusted p-values. Error bars indicate SD.
Fig 3: Dynein associates with TRIM25 and with the proteasome.(A) MCF7 cells where TRIM25 was knockout cells with 2 different gRNAs (KO1, KO2) and a non-targeting control gRNA (NT) were lysed. An aliquot of the lysate was used to monitor p300 and dynein levels. Immunodetection of the proteasomal protein α7 was performed for loading control. From 3.2 mg of the remaining lysate, p300 was precipitated, and associated Dynein was monitored by Western blotting. (B) MCF7 cells where TRIM25 was knocked out by CRISPR/Cas9 (KO) and a non-targeted control (NT) were plated in chamber slides. PLA was performed using antibodies against p300 and dynein or against p300 and mouse IgG or against dynein and rabbit IgG. Nuclei were visualized by DAPI (blue), the PLA signals were labelled with Texas red (red). Images were obtained using a Zeiss LSM-900 confocal microscope. The dots of 78 non-targeted cells and of 87 knock-out cells were counted. Mean values and standard deviations were plotted. (**: P < 0.01). (C) MCF7 cells were lysed. An aliquot of the lysate was used to monitor dynein and TRIM25 levels. From 5 mg of the lysate, TRIM25 was precipitated. Two-thirds of the precipitate were loaded onto one gel, blotted, and associated dynein was monitored by Western blotting. The remaining third was loaded onto a second gel, blotted, and probed for TRIM25. (D) MCF7 cells were lysed. An aliquot of the lysate was used to monitor dynein, α7, and TRIM25 levels. From 5 mg of the lysate, dynein was precipitated. Two-thirds of the precipitate were loaded onto one gel, blotted, and associated TRIM25 and α7 were monitored by Western blotting. The remaining third was loaded onto a second gel, blotted, and probed for dynein. (E) Non-targeted and TRIM25 Knock-out MCF7 cells were lysed. Aliquots of the lysate were used to monitor dynein and α7 and TRIM25 levels. Immunodetection of α7 was performed for loading control. From 3.8 mg of the lysates, dynein was precipitated. Associated α7 was monitored by Western blotting. (F) MCF7 cells where TRIM25 was knocked-out by CRISPR/Cas9 (KO) and a non-targeted control (NT) were plated in chamber slides. PLA was performed using antibodies against p300 and α7 or against p300 and mouse IgG. Nuclei were visualized by DAPI (blue) and the PLA signals were labelled with Texas red (red). Images were obtained using a Zeiss LSM-900 confocal microscope. The dots of 200 non-targeted cells and of 236 knock-out cells were counted. Mean values and standard deviations were plotted. (****: P < 0.001).Source data are available for this figure.
Fig 4: Ba-Exo promotes Mφ phagocytosis. The phagocytosis of 3D4/21 cells were measured by the quantitative uptake of fluorescein isothiocyanate dextran (FITC-dextran) after Ba-Exo and Exo pretreatment. (A), The quantitative uptake of FITC-dextran was detected by laser-scanning microscope. Representative images were captured under a laser scanning confocal microscope (Zeiss LSM900). (B), The quantitative uptake of FITC-dextran was detected by BD FACSCanto™ flow cytometer.
Fig 5: Effects of FAK on expression levels of PI4KIIα and PI4KIIIβ in HeLa cells.A, expression levels of PI4KIIα and PI4KIIIβ were detected via Western blot. Equal proteins were subjected to 7.5% SDS-PAGE gel and verified by indicated antibodies. α-Tubulin was used as a loading control. Data were quantified using Image J software and obtained from three independent experiments. All values reflect one-way ANOVA with Tukey’s post hoc analysis as the mean ± SD. ∗∗p < 0.01; ∗∗∗p < 0.001. B, the mRNA levels of PI4KIIα and PI4KIIIβ in the WT, FAK KO, and Res HeLa cells were detected by qPCR. GAPDH was used as an internal control. All values were normalized to that of the GAPDH. The ratio of the intensities of WT was set as 1.0. Data represent the mean ± SD from three independent experiments. p > 0.05; ∗p < 0.05; ∗∗p < 0.01. C, the expression vector containing GFP-PI4KIIα was transfected into the WT, FAK KO, or Res HeLa cells. After transfection for 72 h, the fluorescence for PI4KIIα expression was detected using a ZEISS LSM 900 confocal microscope. Scale bars, 10 μm. D, the expression levels of endogenous PI4KIIIβ were detected by immunostaining with an anti-PI4KIIIβ antibody. Scale bars, 10 μm. ns, no significance.
from Carl Zeiss Microscopy for ZEISS LSM 900 with Airyscan 2