Journal: iScience

Article Title: Epithelial cell expansion drives cyst progression in genetic models of autosomal recessive polycystic kidney disease

doi: 10.1016/j.isci.2026.116288

Figure Lengend Snippet: Lineage tracing cholangiocytes in cyst growth in Pkhd1 CreER/CreER ; ZS-Green rats (A and B) Schematic drawing of the experimental design, individual cells were labeled with ZSGreen at the dilated tubule or early cystic stage and examined the signal at later advanced cystic stages. (C) Immuno-fluorescence of anti-CK19 (red) on liver sections of Pkhd1 CreERl+ ; ZS-Green and Pkhd1 CreERlCreER ; ZS-Green rats at P7 induced with tamoxifen intraperitoneally injected with a dose of 200 mg/kg into experiment rats at P5. (D and E) Images of liver sections of Pkhd1 CreER/+ ; ZS-Green (D) ( n = 3) and Pkhd1 CreER/CreER ; ZS-Green (E) ( n = 3) rats at P90 induced with tamoxifen at P5. (F) Aggregated data on the number of ZSGreen cells in continuous stretch in the bile ducts of Pkhd1 CreER/+ ; ZS-Green ( n = 44) and cysts of Pkhd1 CreER/CreER ; ZS-Green ( n = 47) liver. Continuous stretch from 3 Pkhd1 CreER/+ ; ZS-Green and 3 Pkhd1 CreER/CreER ; ZS-Green rats. (G) Immuno-fluorescence with anti-CK19 (red) antibody on liver sections of Pkhd1 CreER/+ ; ZS-Green and Pkhd1 CreER/CreER ; ZS-Green rats at P30, P60, and P90 induced with tamoxifen at P5. (H and I) Immuno-fluorescence using anti-EdU and anti-CK19 antibodies (H) and aggregated data of the ratio of EdU positive cells in the bile duct cells (I) on the section of Pkhd1 CreER/+ and Pkhd1 CreER/CreER rats injected with EdU 3 h before sacrifice. Error bars represent mean ± s.e.m, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by Student’s t test (F and I). Scale bars, 400 μm (C); 200 μm (D, E, and G); 100 μm (H).

Article Snippet: Rosa Brainbow (stock #: 017492), CAG CreERT2 (stock #: 004682), and Ck19 CreER (stock #: 026925) were ordered from Jackson Laboratory.

Techniques: Labeling, Fluorescence, Injection

Journal: iScience

Article Title: Epithelial cell expansion drives cyst progression in genetic models of autosomal recessive polycystic kidney disease

doi: 10.1016/j.isci.2026.116288

Figure Lengend Snippet: Lineage tracing cholangiocytes in cyst growth in a mouse model of ARPKD using Rosa Brainbow mice (A and B) Schematic drawing of the experimental design, individual cells were labeled with distinct fluorophores at the early cystic stage, and the fluorescence was examined at a more advanced cystic stage. (C) Confocal images with GFP, YFP, BFP, and RFP channels of the liver section of CK19 CreER ; Pkhd1 +/− ; Rosa Brainbow/+ and CK19 CreER ; Pkhd1 −/− ; Rosa Brainbow/+ mice at P30 with a single dose of tamoxifen at P28. (D) Confocal images with GFP, YFP, BFP, and RFP channels of the liver section of CK19 CreER ; Pkhd1 +/− ; Rosa Brainbow/+ and CK19 CreER ; Pkhd1 −/− ; Rosa Brainbow/+ mice at P90 with a single dose of tamoxifen at P28. (E and F) Clonal number (E) and clonal frequency (F) of each fluorophore in CK19 CreER ; Pkhd1 +/− ; Rosa Brainbow/+ and CK19 CreER ; Pkhd1 −/− ; Rosa Brainbow/+ mice at P90, data were collected from 5 mice of each genotype. Error bars represent mean ± s.e.m, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by two-way ANOVA (F). Scale bars, 50 μm (C); 100 μm (D).

Article Snippet: Rosa Brainbow (stock #: 017492), CAG CreERT2 (stock #: 004682), and Ck19 CreER (stock #: 026925) were ordered from Jackson Laboratory.

Techniques: Labeling, Fluorescence

Journal: iScience

Article Title: Epithelial cell expansion drives cyst progression in genetic models of autosomal recessive polycystic kidney disease

doi: 10.1016/j.isci.2026.116288

Figure Lengend Snippet: Examine cystogenic potential of individual Pkhd1 homozygous cells in the background of heterozygous cells in Pkhd1 CreER/fl rat, and model cyst growth dynamics (A) Schematic drawing of the experimental design of analysis, the green labeled cell represents Pkhd1 CreER/- ; ZsGreen cell to assay the cystogenic potential of individual Pkhd1 homozygous cells. (B and C) Hematoxylin and eosin staining of the 1-year-old rat kidney sections (B) and aggregated kidney body weight ratio (C) of Pkhd1 CreER/fl rat induced with corn oil ( n = 6) or tamoxifen ( n = 7). (D and E) Masson-Trichrome staining of 1-year-old rat liver sections (D) and the liver body weight ratio (E) of Pkhd1 CreER/fl ; ZS-Green induced with corn oil ( n = 6) or tamoxifen ( n = 7). (F) Immuno-fluorescence with anti-Aqp2 (red) antibody on the kidney section of Pkhd1 CreER/fl ; ZS-Green rats at 1-month-old induced with tamoxifen at P5 and P6. (G) Immuno-fluorescence with anti-Ck19 (red) antibody on liver sections of Pkhd1 CreER/fl ; ZS-Green rats at 1-month-old induced with tamoxifen at P5 and P6. (H) Kidney section of a 12-month-old Pkhd1 CreER/fl ; ZS-Green rat induced with corn oil or tamoxifen at P5 and P6. (I) Immuno-fluorescence with anti-Aqp2 (red) antibody on kidney sections of 12-month-old Pkhd1 CreER/fl ; ZS-Green rat and counterstained with DAPI, induced with corn oil or tamoxifen at P5 and P6. (J) Liver sections of 12-month-old Pkhd1 CreER/fl ; ZS-Green rat induced with corn oil or tamoxifen at P5 and P6. (K) Immuno-fluorescence with anti-CK19 antibody (red) on liver sections of 12-month-old Pkhd1 CreER/fl ; ZS-Green rat and counterstained with DAPI induced with corn oil or tamoxifen at P5 and P6. (L) Mathematic formula of cyst size based on cell expansion and cell doubling time. (M) Plot a graph of cyst size by time based on the doubling time of 20 days and 30 days from a single cell. The dots on the right represent the measured diameter of liver cysts of 12-month-old Pkhd1 CreER/fl ; Zs-Green rats induced with tamoxifen at P5 and P6 ( H). Error bars represent mean ± s.e.m, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by Student’s t test (C and E). Scale bars, 2 mm (B); 1 mm (D); 200 μm (F, G, I, and K); 800 μm (H and J).

Article Snippet: Rosa Brainbow (stock #: 017492), CAG CreERT2 (stock #: 004682), and Ck19 CreER (stock #: 026925) were ordered from Jackson Laboratory.

Techniques: Labeling, Staining, Fluorescence, Single Cell

Journal: iScience

Article Title: Epithelial cell expansion drives cyst progression in genetic models of autosomal recessive polycystic kidney disease

doi: 10.1016/j.isci.2026.116288

Figure Lengend Snippet: Pharmacological inhibition of Cdk1 with RO-3306 slows fibrocystic liver disease in Pkhd1 −/− mice (A) Expression of Cdk1 in HEK293T cells treated with different concentrations of RO-3306 via western blot. (B) Cdk1 expression in the liver, kidney, and spleen of mice treated with DMSO or RO-3306 via western blot. The organs were harvested at 3, 6, 9, and 12 h after the administration of four-week-old mice intraperitoneally injected with RO-3306 at a dosage of 20 mg/kg. (C) Schematic drawing of the treatment of Pkhd1 mutant mice with DMSO or RO-3306. (D and E) Immuno-fluorescence with anti-Ck19 antibody on liver sections (D) and aggregated data of cystic index defined by Ck19 circled area per parenchyma captured (E) of WT ( n = 5), Pkhd1 −/− mutant treated with DMSO ( n = 9, male: symbol with color; female: symbol with gray), and Pkhd1 mutant mice treated with RO-3306 ( n = 9, male: symbol with color; female: symbol with gray) from P28 to P90. (F and G) Masson-Trichrome staining of liver sections (F) and aggregated data of the ratio of Masson staining positive area in the parenchyma captured (G) of the WT ( n = 5), Pkhd1 −/− mutant treated with DMSO ( n = 9, male: symbol with color; female: symbol with gray), and Pkhd1 mutant mice treated with RO-3306 ( n = 9, male: symbol with color; female: symbol with gray) from P28 to P90. (H and I) Immuno-fluorescence of anti-CK19 (green) and anti-Ki67 (red) (H) and aggregated data of the ratio of Ki67+Ck19+ cells in CK19+ cells (I) on the liver sections of WT ( n = 3), Pkhd1 −/− mice treated with DMSO ( n = 3), and Pkhd1 −/− mice treated with RO-3306 ( n = 3). (J and K) Analyzing the expression of PCNA (J) via western blot and aggregated data of the densitometric ratios of PCNA to Hsp90 (K) in WT ( n = 3), Pkhd1 −/− mice treated with DMSO ( n = 3), and Pkhd1 −/− mice treated with RO-3306 ( n = 3). (L and M) TUNEL (red) staining counter stained with anti-CK19 antibody (L) on the liver sections and aggregated data of the ratio of TUNEL+ cells within in CK19+ cells (M) on the liver sections of WT ( n = 3), Pkhd1 −/− mice treated with DMSO ( n = 3), and Pkhd1 −/− mice treated with RO-3306 ( n = 3). (N and O) Analysis of the expression of cleaved-Caspase 3 (N) and Hsp90 via western blot and the densitometric ratios of cleaved-Caspase 3 to Hsp90 (O), which were normalized to the ratio of the WT group. (P) Analyzing the expression of CDK1, α-SMA, and PDGFβ in WT ( n = 3), Pkhd1 −/− treated with DMSO ( n = 3), and Pkhd1 −/− treated with RO-3306 ( n = 3) livers via western blot. (Q, R, and S) Densitometric ratios of Cdk1 (Q), PDGFRβ (R), and α-SMA (S) to Hsp90 for in (P), which were normalized to the ratio of the WT group. (T) Immuno-fluorescence of anti-COL3a, anti-α-SMA, and anti-PDGFβ antibodies on the liver sections of WT, Pkhd1 −/− mice treated with DMSO, and Pkhd1 −/− mice treated with RO-3306. Error bars represent mean ± s.e.m, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by two-way ANOVA. Scale bars, 200 μm (D and T); 1 mm (F); 100 μm (H and L).

Article Snippet: Rosa Brainbow (stock #: 017492), CAG CreERT2 (stock #: 004682), and Ck19 CreER (stock #: 026925) were ordered from Jackson Laboratory.

Techniques: Inhibition, Expressing, Western Blot, Injection, Mutagenesis, Fluorescence, Staining, TUNEL Assay