Journal: Cell death discovery

Article Title: Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys.

doi: 10.1038/s41420-024-02131-y

Figure Lengend Snippet: Fig. 1 Generation of doxycycline (Dox)-inducible SV40LT in human kidney CD10+ proximal tubule epithelial cells (PTECs) and PDGFRβ+

Article Snippet: CD10+/CD31−/CD45−/dapi- PTECs were sorted using a SONY SH800 sorter (Sony Biotechnology, San Jose, CA, USA) (Supplementary Fig. S1).

Techniques:

Journal: Cell death discovery

Article Title: Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys.

doi: 10.1038/s41420-024-02131-y

Figure Lengend Snippet: Fig. 4 Functional analysis of human kidney CD10+ PTECs and PDGFRβ+ cell lines with doxycycline-inducible SV40LT expression and crosstalk between PTECs and fibroblasts. A, B Fluorescence intensity of CD10+ PTECs with and without efflux transport blockers. The cells were first incubated with the substrates calcein-AM (1 μM) and CMFDA (1.25 μM), after which the efflux transport blockers PSC833 (5 μM) and MK571 (5 μM) were added. n = 9–18, **P < 0.01, ****P < 0.0001, two-tailed unpaired t test. C, D Gene expression of COL1A1 and FN1 in human kidney PDGFRβ+ cells exposed to IL-1β (100 ng/ml) or TGFβ (10 ng/ml). *P ≤0.05, **P < 0.01, ****P < 0.0001; two-tailed unpaired t test. N = 6. E Cytokine arrays incubated with pooled supernatant from senescent (−dox) and nonsenescent (+dox) PTECs (n = 3 per condition was pooled and added to one membrane). F Quantification of (E). G Mass spectrometry signal of MMP-7 in cell culture supernatant from nonsenescent (dox+) and senescent (dox−) PTECs. N = 4. H Schematic representation of the coculture experiments. F Supernatants from day 5 senescent CD10+ PTECs were transferred to PDGFRβ+ cells for 3 days. I Gene expression of ACTA2, COL1A1, and FN1 in PDGFRβ+ cells. *P ≤0.05, two- tailed unpaired t-test. N = 5–6. Mean values are shown ± SD. J Immunofluorescence staining of αSMA and Col1a1 in PDGFRβ+ cells. Scale bars, 50 μm. N = 3.

Article Snippet: CD10+/CD31−/CD45−/dapi- PTECs were sorted using a SONY SH800 sorter (Sony Biotechnology, San Jose, CA, USA) (Supplementary Fig. S1).

Techniques: Functional Assay, Expressing, Fluorescence, Incubation, Two Tailed Test, Gene Expression, Membrane, Mass Spectrometry, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: Plk1 promotes renal tubulointerstitial fibrosis by targeting autophagy/lysosome axis

doi: 10.1038/s41419-023-06093-4

Figure Lengend Snippet: A mPTC cells were transfected with Plk1 shRNA and stimulated with TGF-β1. FN and LC3 were analyzed by western blot. n = 3-5. B , C mPTC cells were treated with different concentrations of BI6727 and stimulated with TGF-β1. FN, Smad2, p-Smad2, LC3 and P62 were analyzed by western blot. n = 3-4. *compared to ctr group, # compared to TGF-β1 group. D Intralysosomal pH detection by lysosensor dye in mPTC cells treated with BI6727 in the presence or absence of TGF-β1. Scale bar=20 µm. n = 3. E Western blot analysis of FN in mPTC cells treated with CQ in response to TGF-β1. n = 3. F Western blot analysis of FN in mPTC cells when ATP6V1A is knocked down in response to TGF-β1. n = 3. G Western blot analysis of CyclinB1 and pH3 when ATP6V1A was knocked down. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. # P < 0.05, ## P < 0.01, ### P < 0.001.

Article Snippet: Rat kidney fibroblast cells NRK49F or mouse primary tubular epithelial cells mPTC were purchased from ATCC and were cultured at 37 °C under 5% CO 2 in DMEM or DMEM/F12 medium containing 10% FBS supplemented with penicillin-streptomycin (100 IU/mL and 100 mg/mL).

Techniques: Transfection, shRNA, Western Blot