Journal: Advanced Science

Article Title: Deciphering the Impact of RAC1‐SPTAN1 in ARPKD Cystogenesis Using Multifaceted Models

doi: 10.1002/advs.202524001

Figure Lengend Snippet: Recreating ARPKD pathophysiology using a microfluidic organoid‐on‐chip model. (A) Schematic representation of a novel organoid‐on‐chip model utilizing a microfluidic chip. PSC, pluripotent stem cell; NPC, nephron progenitor cell; NPO, nephron organoid. (B) Upper panels: Whole‐organoid 3D confocal imaging stacks of fluidic‐cultured PKHD1 + / − and PKHD1 − / − organoids on day 35. Lower panels: Imaris surface for CDH1. The yellow signal observed outside corresponds to non‐specific background from Geltrex. Scale bars: 200 µm. (C) Quantification of the total tubule volume in nephron organoids on day 35. The left panel shows the results for CDH1+ tubules. Each dot represents the value of a single organoid. Each condition contains 5–9 organoids. ** p < 0.01, * p < 0.05, n.s.: not significant. (D) Immunostaining for the ciliary marker TUBA1A in fluidic‐cultured PKHD1 + / − and PKHD1 − / − organoids on day 35. Asterisks indicate dilated lumens, while arrowheads mark primary cilia. Scale bars: upper panels, 50 µm; lower panels, 10 µm. (E) Immunostaining for the ciliary marker TUBA1A and the basolateral marker ATP1A1 in fluidic‐cultured kidney organoids on day 35. Asterisks indicate apical side. Scale bars: upper panels, 50 µm; lower panels, 10 µm. (F) Live perfusion imaging of 3kDa‐Dextran under pulsatile flow conditions (0.4 and 0.04 mL min −1 every 0.5 s). The lumens of WGA+ tubules are filled with dextran, consistent with tubular luminal flow. Scale bar: 100 µm.

Article Snippet: The sections were incubated overnight at 4°C with following primary antibodies/reagent: SLC8A1 (1:25; MA3‐926, Invitrogen), ATP1A1 (1:50; A5‐DSHB, DSHB), and FITC‐conjugated LTL (1:100; FL‐1321‐2, Vector).

Techniques: Imaging, Cell Culture, Immunostaining, Marker

Journal: Advanced Science

Article Title: Deciphering the Impact of RAC1‐SPTAN1 in ARPKD Cystogenesis Using Multifaceted Models

doi: 10.1002/advs.202524001

Figure Lengend Snippet: SLC8A1‐positive connecting tubules are the potential origin of ARPKD cysts. (A) UMAP generated from scRNA‐seq of 24 healthy human kidneys. Po, podocytes; PT, proximal tubules; LoH, loop of Henle; DCT, distal convoluted tubules; CNT, connecting tubules; CD‐PC, collecting duct principal cells; IC, intercalated cells; FB, fibroblast cells; EC, endothelial cells; VSM, vascular smooth muscle cells; ImC, immune cells. (B) PKHD1 and signature gene expression of each nephron segment. (C) Immunostaining for SLC8A1 in SPTAN1 + / + and SPTAN1 + / − organoids. Upper panels, static condition; lower panels, flow condition. Scale bars: 100 µm. (D) Quantification of the short‐axis diameters of CDH1+ tubules. Each dot represents the mean value of each organoid. Each condition contains 5 organoids. * p < 0.05, n.s.: not significant. (E) Immunostaining for SLC8A1 in fluidic‐cultured PKHD1‐/‐ organoids on day 35. Scale bar: 500 µm. (F) Quantification of the short‐axis diameters of CDH1+ tubules in PKHD1 − / − organoids. Each dot represents the measurement of an individual tubule. Each condition contains 30 and 18 tubules respectively from 2 organoids. **** p < 0.0001. (G) Immunostaining for SLC8A1 and ATP1A1 in human kidneys of healthy controls and ARPKD patients. Asterisks indicate dilated lumens. Scale bars: 100 µm. (H) Quantification of the SLC8A1 intensity in human samples. Each dot represents the value of a single tubule. Each segment contains 10 tubules or 20 cysts. **** p < 0.0001. PT, proximal tubule; DT, distal tubule; CNT, connecting tubule; CCD, cortical collecting duct; IMCD, inner medullary collecting duct.

Article Snippet: The sections were incubated overnight at 4°C with following primary antibodies/reagent: SLC8A1 (1:25; MA3‐926, Invitrogen), ATP1A1 (1:50; A5‐DSHB, DSHB), and FITC‐conjugated LTL (1:100; FL‐1321‐2, Vector).

Techniques: Generated, Gene Expression, Immunostaining, Cell Culture

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 interacts with RBBP4 in the nucleus. ( A ) The subcellular location of KTN1-AS1 in esophageal squamous cell carcinoma (ESCC) cell lines. ( B ) RNA pull-down assay was performed in Kyse150 and Kyse170 cells, and the RNA-related proteins were determined with SDS-PAGE gel and coomassie brilliant blue staining. Original gel image was presented in Supplementary Fig. A. ( C ) Western blot assay was performed to detect the specific association between RBBP4 and KTN1-AS1 in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. B, with blots cut prior to hybridization with antibodies. ( D ) RIP assay showed the interaction between KTN1-AS1 and RBBP4 in Kyse150 and Kyse170 cells. ( E ) The regulation effect of KTN1-AS1 on RBBP4 expression was detected by qRT-PCR and western blot. Original western blots were presented in Supplementary Fig. C, with blots cut prior to hybridization with antibodies. ( F ) The relevance between KTN1-AS1 and RBBP4 expression was predicted by the GEPIA database. ( G ) The relative expression of RBBP4 in 182 tumor samples compared with 286 normal samples obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) database. ( H ) The expression levels of RBBP4 in ESCC and the corresponding normal tissues. ( I ) The expression levels of RBBP4 in ESCC cell lines. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Pull Down Assay, SDS Page, Staining, Western Blot, Hybridization, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 relates to epithelial-to-mesenchymal transition (EMT) process by interacting with RBBP4 and HDAC1 to silence E-cadherin expression. ( A ) The mRNA expression levels of E-cadherin , N-cadherin , Vimentin , and MMP2 after KTN1-AS1 overexpression and knockdown. ( B ) The regulatory effect of KTN1-AS1 on protein levels of E-cadherin, N-cadherin, Vimentin, and MMP2 was detected by western blot. Original western blots were presented in Supplementary Fig. D, with blots cut prior to hybridization with antibodies. ( C ) Inhibition of RBBP4 increased the expression level of E-cadherin and partially reversed the regulation effect of KTN1-AS1 on the expression level of E-cadherin in Kyse150 and Kyse170 cells. ( D ) Co-IP assay was performed to examine the RBBP4-HDAC1 interaction in the groups with KTN1-AS1 overexpression and inhibition in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. E, with blots cut prior to hybridization with antibodies. ( E ) RIP assay showed the interaction between KTN1-AS1 and HDAC1 in Kyse150 and Kyse170 cells. ( F ) After transfection of pcDNA3.1-NC or pcDNA3.1-KTN1-AS1 for 12–24 h in Kyse150 and Kyse170 cells, then cells with pcDNA3.1-KTN1-AS1 were treated with or without 300 nM Trichostatin A (TSA) for additional 48 h, the mRNA expression of E-cadherin was detected by qRT-PCR method. ( G ) ChIP-qPCR was performed to detect the enrichment of ac-H3 in the promoter region of E-cadherin after overexpression and knockdown of KTN1-AS1 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Hybridization, Inhibition, Co-Immunoprecipitation Assay, Transfection, Quantitative RT-PCR, ChIP-qPCR

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: RBBP4 partially reverses the biological function of KTN1-AS1 on esophageal squamous cell carcinoma (ESCC) cells. ( A , B ) MTS and clone formation assays were performed to analyze the cell proliferation ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. ( C , D ) Wound healing and transwell invasion assays were conducted to explore the migration and invasion ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Transfection, Migration