Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Podocytes exhibit high levels of basal autophagy and autophagic flux. (A) Representative images obtained from cryosections of 4-month old Gfp-Lc3 mice stained for the basement membrane marker NID1/nidogen-1/entactin (red) and GFP-LC3 (green). (B) Two-photon images of glomeruli from Gfp-Lc3 mice perfused with dextran as a marker for glomerular capillaries (in red) displaying autophagosomes in vivo (in green, marked with arrow). (C) Representative images obtained from cryosections of 4-month-old Rfp-Gfp-Lc3 mice stained for NID1 (purple). RFP-LC3 and GFP-LC3 fluorescence is endogenous. (D) Quantification of (C) out of 30 glomeruli from 3 mice each with surrounding tubular cells (** ≤ 0.01, * ≤ 0.05). (E) Cryosections displaying glomeruli (upper panel) and tubular system (lower panel) stained for NID1 (red) and GFP-LC3 (anti-GFP antibody, green) in 4-month-old WT mice with and without chloroquine (4 h after chloroquine [Cq] administration i.p. 100 mg Cq/kg BW). (F) Western blot from immortalized human podocyte cell line and proximal tubular cell line (HK2) for MTORC1 downstream targets and LC3 abundance. (G) Densitometry obtained from (F) (** ≤ 0.01)

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Staining, Marker, In Vivo, Fluorescence, Western Blot

Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Basal autophagy is independent of MTOR activity in podocytes in vivo . (A) Schematic of generating podocyte-specific deletion of Rptor or Tsc1 using Nphs2-Cre mice and Cre-Lox technique. (B) Cryosections from 2-week-old mice bearing podocyte-specific knockout for Rptor and transgenic for Gfp-Lc3 compared to Gfp-Lc3 WT mice (NID1 in red, GFP-LC3 in green). (C) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 3 mice (ns, not significant). (D) Cryosections from 2-week-old mice bearing podocyte-specific knockout for Tsc1 and transgenic for Gfp-Lc3 compared to Gfp-Lc3 WT mice (NID1 in red, GFP-LC3 in green). (E) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 3 mice (ns, not significant). (F) Western blot out of glomerular lysates obtained from 2-week-old mice for MTORC1 downstream targets and LC3 and SQSTM1 abundance. (G) Densitometry for LC3-II, SQSTM1 and p-RPS6 obtained from 3 WT glomerular lysates and 3 glomerular lysates obtained from 2-week-old mice bearing a podocyte-specific deletion of Rptor or Tsc1 , respectively (** ≤ 0.01, * ≤ 0.05, ns, not significant)

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Activity Assay, In Vivo, Knock-Out, Transgenic Assay, Western Blot

Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Effects of acute and long-term pharmacological inhibition of MTORC1 activity on autophagy. (A) Schematic of the short-term treatment regimen (rapamycin vs. vehicle, n = 5 each, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (B) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 5 mice per group (vehicle vs. rapamycin). (C) Representative cryosections from 16-week-old mice transgenic for GFP-LC3 with and without rapamycin treatment (NID1 in red, GFP-LC3 in green, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (D) Representative western blot from glomerular lysates from rapamycin and vehicle treated mice (WT mice, n = 3 each, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (E) Densitometric quantification of (D) (** ≤ 0.01, * ≤ 0.05). (F) Schematic of the long-term treatment regimen (rapamycin dose: 4 mg/kg BW i.p. for 3 weeks). (G) Quantification of serum levels of rapamycin (n = 6 each) (*** ≤ 0.001). (H) Representative western blot from glomerular lysates from rapamycin long-term, short-term and vehicle treated mice. (I) Densitometry from 3 glomerular lysates of each treatment and vehicle controls for LC3-II, SQSTM1 and p-RPS6 Ser235/236 (** ≤ 0.01, ns, not significant). LT, long-term; ST, short-term

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Inhibition, Activity Assay, Transgenic Assay, Western Blot