PTH1R/PTHR1 Antibody (4D2) NBP1-51640 from Novus Biologicals, a Bio-Techne Brand

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Scientific reports
November 26 2019

Fig 1: Generation of functional kidney spheroids using immortalized mouse primary cells. (a) Schematic diagram of SFU protocol (combination of hanging drop and rotation) and maturation factors for generation of kidney spheroids for structural maturation. (b) Quantitative RT-PCR analysis of kidney-specific segment markers in mouse kidney spheroids (mKidS) compared with 2D controls (2D CTL) and mouse kidney (mKidney). n = 9 for 2D CTL and mKidS, n = 3 for mKidney. (c) Western blotting analysis for renal multi-lineage markers in mouse kidney spheroids compared with 2D CTL and mKidney. The grouping of gels/blots cropped from different parts of the same gel. (d) Immunostaining images for cell polarity of Zo-1, an apical membrane, and Na/K+ ATPase, a basolateral membrane marker in mKidS. Scale bar, 10 µm. (e) Representative images of mKidS at day 5 of maturation stained with hematoxylin and eosin (H&E) or immunostained for the kidney-specific markers LTL, PNA, AQP2, and nephrin; transmission electron microscopy of kidney spheroids displaying long (black arrowhead) and short (red arrowhead) villi with tight junctions (black arrow) and foot processes (asterisks in the middle panel). Scale bar of TEM image, 1 µm. Mouse kidney from 12 weeks-old mice was used as a positive control for immunostaining of mKidS. (f) Response to parathyroid hormone (PTH) compared with 2D cells and 3D control spheroids (without maturation factors, CTL). n = 6 for 2D CTL, 2D PTH treated, n = 10 for 3D CTL and mKidS. (g) GGT activity in mKidS compared to 3D CTL after 2 and 4 days of maturation. n = 8 for CTL and mKidS, respectively. (h) Temperature-dependent albumin-fluorescein uptake compare to 3D CTL. n = 12 for 3D CTL and mKidS, respectively. (I,j) Relative mRNA expression of apical (i) and basolateral (j) transporters in mKidS compared with 2D cultured cells, TCMK-1 spheroids generated using the same protocol for mKidS (TCMK-3D) and mKidney. n = 8 for 2D CTL and mKidS and n = 4 for TCMK-3D. (k) Representative images of mKidS immunostained for p-GP, OCT2, and PTHR. All data are shown as the means ± s.e.m. *p < 0.05 compared with 2D or 3D control by unpaired Student’s t-test. Scale bar, 20 µm.
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Scientific reports
November 26 2019

Fig 2: Generation of functional kidney spheroids using immortalized human primary cells. (a) Quantitative RT-PCR analysis of kidney-specific segment markers in hKidS compared with 2D controls (2D CTL) and human kidney lysates (hKidney). n = 8 for 2D CTL and hKidS and n = 3 for hKidney. (b) Western blotting analysis for renal multi-lineage marker in mouse kidney spheroids compared to 2D CTL and hKidney. The grouping of gels/blots cropped from different parts of the same gel. (c) Representative images for cell polarity of Zo-1, an apical membrane, and Na/K ATPase, a basolateral membrane marker in hKidS. Scale bar, 10 µm. (d) Representative images of human kidney spheroids (hKidS) at day 5 of maturation stained for the kidney-specific markers LTL, PNA, AQP2, and Nephrin. Human kidney was used as positive control for immunostaining of hKidS. (e) Responses to parathyroid hormone (PTH) of hKidS compared with 3D control spheroids (without maturation factors, CTL) and 2D cells. (f) GGT activity in hKidS compared with 3D CTL. (g) Temperature-dependent albumin-fluorescein uptake of hKidS compared to 3D CTL. n = 10 for 3D CTL and hKidS. (h,i) Relative mRNA expression of apical (h) and basolateral (i) transporters in hKidS compared to 2D cultured cells, hPTECs spheroids generated using the same protocol for hKidS (hPTECs-3D), and hKidney. n = 9 for 2D CTL and hKidS and n = 3 for hPTECs-3D and hKidney. (j) Representative images of hKidS immunostained for p-GP, OCT2, and PTHR. All data are shown as the means ± s.e.m. *p < 0.05 compared with 2D or 3D control by unpaired Student’s t-test. Scale bar, 20 µm.