Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: Disruption of LAP impairs POS degradation by the RPE. RPE-J cells were transfected with Scrambled (Scr), Rubcn (A), Atg5 (B), Becn1 (C), Atg4b (D), Rb1cc1 (E) or Atg14 (F) siRNA oligonucleotides. Cells were then fed unlabeled POS for 6 h, washed extensively with PBS-CM to remove unbound POS and then examined at 16 h or 24 h. Cells were also fed unlabeled POS for 6 h, washed with PBS-CM and then treated with the lysosomal protease inhibitors E64d (10 µM) and pepstatin A (10 µM) (E/P) and examined at 16 h (A-F). POS degradation by RPE-J cells was determined by measuring the levels of RHO (rhodopsin) monomers by western blot analysis. ACTB was the loading control. Representative blots from 3 independent experiments are shown. (G-H) Following knockdown of Rubcn with siRNA oligonucleotides RPE-J cells were fed labeled POS (red) and stained for LAMP1 (green) (G) or LAMP2 (green) (H). The percentage (%) of POS phagosomes that colocalized with LAMP1 (G, top left micrograph, arrows) or LAMP2 (H, bottom left micrograph, arrows) were quantified by confocal microscopy 16 h after initial POS feeding. Arrowheads identify POS phagosomes not associated with LAMP1 (G, top right micrograph) or LAMP2 (H, bottom right micrograph). Bar graphs quantification represent the average of 3 independent experiments; *p < 0.05.

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: Disruption, Transfection, Western Blot, Control, Knockdown, Labeling, Staining, Confocal Microscopy

Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: In vitro starvation prevents optimal POS degradation by the RPE. (A) RPE-J cells were cultured in complete medium (DMEM with 4% FBS) or starvation medium (DMEM with 0.1% FBS) for 24 h, stained for MAP1LC3B (LC3B, green) and DAPI (nuclei; blue) and then analyzed by confocal microscopy. Representative confocal images from 2 independent experiments are shown. (B) RPE-J cells cultured in complete medium or starvation medium were fed unlabeled POS for 6 h, extensively washed with PBS-CM to remove unbound materials and then examined at 16 h or 24 h. Cells were also fed unlabeled POS for 6 h, washed with PBS-CM and then incubated in complete medium or starvation medium with the lysosomal protease inhibitors E64d (10 µM) and pepstatin A (10 µM) (E/P) and examined at 16 h. The expression level of RHO monomers was determined by western blot. ACTB was the loading control. Representative blots from 3 independent experiments are shown. (C) RPE-J cells cultured in complete medium or starvation medium were fed labeled POS for 6 h. The number of total POS and internalized POS per RPE cell shown in bar graphs were quantified using confocal microscopy and averaged from 3 independent experiments. (D) RPE-J cells cultured in complete medium or starvation medium were fed labeled POS (red) and stained for RUBCN (green) and DAPI (blue). The percentage (%) of RUBCN+ phagosomes (arrows, left micrograph, inset) were quantified by confocal microscopy 16 h after initial POS feeding. Arrowheads identify POS phagosomes (red) not associated with RUBCN (right micrograph, inset). Bar graphs quantification represent the average of 3 independent experiments; *p<0.05. (E) RPE-J cells cultured in complete medium or starvation medium were fed labeled POS (red) and stained for LAMP1 (green) (left micrographs) or LAMP2 (green) (right micrographs). The percentage (%) of POS phagosomes that colocalized with LAMP1 (top left micrograph, arrows, inset) or LAMP2 (top right micrograph, arrows, inset) were quantified by confocal microscopy 16 h after initial POS feeding. Arrowheads identify POS phagosomes not associated with LAMP1 (bottom left micrograph) or LAMP2 (bottom right micrograph). Data quantification shown in bar graphs represent the average of 3 independent experiments; *p < 0.05.

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: In Vitro, Cell Culture, Staining, Confocal Microscopy, Incubation, Expressing, Western Blot, Control, Labeling

Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: RUBCN is highly expressed, while autophagy is suppressed in the morning during active POS phagocytosis. (A) RPE flat mounts were freshly prepared at 7 AM and 7 PM from 16-wk-old C57BL/6J mice to examine the expression of RUBCN (green) and RHO (red). The percentage (%) of RHO+ phagosomes that colocalized with RUBCN (top right micrograph, arrows) was determined by capturing confocal images of 100- × 100-µm fields (4–5 fields per RPE flat mount). Then counting the number of RUBCN+ phagosomes per field of view. Arrowheads identify RHO+ phagosomes not associated with RUBCN (bottom right micrograph). Bar graphs quantification represent the average of 3 independent experiments. Each experiment contained at least 3 mice per time point; *p<0.05. (B) RPE and retinae were isolated at 7 AM and 7 PM from 16-wk-old C57BL/6J mice. Western blots were performed to determine the expression levels of RUBCN, SQSTM1, BECN1, ATG14 and RB1CC1. ACTB was the loading control. Representative blots from 3 independent experiments are shown (n = 3 mice per time point). (C-D) RPE were isolated at 7 AM and 7 PM from 16-wk-old C57BL/6J mice and the expression levels of the MTOR substrate RPS6KB1, p-RPS6K1B (Thr389) (C), BECN1 and p-BECN1 (Ser234) (D) were examined by western blot. Representative blots from 3 independent experiments are shown (n = 3 mice per time point). (E) RPE-J cells were cultured alone or with unlabeled POS for 0.5, 1, 3 or 6 h. The expression levels of RPS6K1B, p-RPS6K1B (Thr389), BECN1 and p-BECN1 (Ser234) were determined by western blot. Representative blots from 3 independent experiments are shown.

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: Expressing, Isolation, Western Blot, Control, Cell Culture

Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: EGFR is involved in autophagy inhibition and the maintenance of RUBCN protein levels during POS phagocytosis. (A) RPE-J cultured with control (DMSO) or erlotinib (2 µM in DMSO) were incubated with media alone or media with POS for 1, 3 or 6 h. The expression levels of RUBCN, SQSTM1, ACTB, RPS6K1B1, p-RPS6K1B1 (Thr389), BECN1 and p-BECN1 (Ser234) were determined by western blots. Representative blot from 3 independent experiments are shown. (B) RPE-J cells cultured with control (DMSO) or erlotinib (2 µM in DMSO) were fed labeled POS (red), stained for RUBCN (green) and DAPI (blue) and analyzed by confocal microscopy. The percentage (%) of POS phagosomes that colocalized with RUBCN (arrows) were quantified by confocal microscopy 16 h after initial POS feeding. Arrowheads identify POS phagosomes not associated with RUBCN. Data quantification shown in bar graphs was performed from confocal images and represent the average of 3 independent experiments; *p < 0.05.

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: Inhibition, Cell Culture, Control, Incubation, Expressing, Western Blot, Labeling, Staining, Confocal Microscopy

Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: RUBCN is required for LAP in the RPE. (A) Following treatment of RPE-J cells with Scrambled (Scr) or Rubcn siRNA oligonucleotides, western blots were performed to assess the expression levels of RUBCN, ATG5, BECN1, ATG4B, ATG14 and RB1CC1. ACTB/β-actin was the loading control. Representative blots from 3 independent experiments are shown. (B) GFP-LC3+ RPE-J cells were transfected with Scr or Rubcn siRNA oligonucleotides and fed labeled POS (red) or they were treated with rapamycin (200 nM). Inset images in top micrographs indicate the recruitment of GFP-LC3 to individual POS phagosomes denoted by arrows (Scr siRNA) or arrowheads (Rubcn siRNA). The scale bar in the inset images is 2 µm. The white squares indicate the location of insets. The percentage (%) of GFP-LC3+ phagosomes (arrows) following POS feeding were quantified following POS feeding from time-lapse confocal images and averaged from 3 independent experiments (top bar graph); *p < 0.05. The number of GFP-LC3+ puncta/cell was quantified 24 h following rapamycin treatment from confocal images and averaged from 3 independent experiments (bottom bar graph).

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: Western Blot, Expressing, Control, Transfection, Labeling

Journal: Autophagy

Article Title: RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

doi: 10.1080/15548627.2017.1380124

Figure Lengend Snippet: Disruption of LAP impairs POS degradation by the RPE. RPE-J cells were transfected with Scrambled (Scr), Rubcn (A), Atg5 (B), Becn1 (C), Atg4b (D), Rb1cc1 (E) or Atg14 (F) siRNA oligonucleotides. Cells were then fed unlabeled POS for 6 h, washed extensively with PBS-CM to remove unbound POS and then examined at 16 h or 24 h. Cells were also fed unlabeled POS for 6 h, washed with PBS-CM and then treated with the lysosomal protease inhibitors E64d (10 µM) and pepstatin A (10 µM) (E/P) and examined at 16 h (A-F). POS degradation by RPE-J cells was determined by measuring the levels of RHO (rhodopsin) monomers by western blot analysis. ACTB was the loading control. Representative blots from 3 independent experiments are shown. (G-H) Following knockdown of Rubcn with siRNA oligonucleotides RPE-J cells were fed labeled POS (red) and stained for LAMP1 (green) (G) or LAMP2 (green) (H). The percentage (%) of POS phagosomes that colocalized with LAMP1 (G, top left micrograph, arrows) or LAMP2 (H, bottom left micrograph, arrows) were quantified by confocal microscopy 16 h after initial POS feeding. Arrowheads identify POS phagosomes not associated with LAMP1 (G, top right micrograph) or LAMP2 (H, bottom right micrograph). Bar graphs quantification represent the average of 3 independent experiments; *p < 0.05.

Article Snippet: The following antibodies were used: RUBCN (21444-1-AP), ATG4B (15131-1-AP), ATG14 (19491-1-AP), RB1CC1/FIP200 (17250-1-AP), SQSTM1/p62 (18420-1-AP) from Proteintech; ATG5 (A0856), LAMP2 (L0668) from Sigma-Aldrich; RHO (MAB5356) from Millipore; BECN1 (3738), p-EGFR (Tyr1068) (3777), RPS6KB1 (2708), p-RPS6KB1 (Thr389) (9234) from Cell Signaling Technology; ACTB-HRP (1615), EGFR (sc-03) from Santa Cruz Biotechnology; MAP1LC3B/LC3B (NB600-1384), p-BECN1 (Ser234) (NBP2-29654) from Novus Biologicals; LAMP1 (ab24170) from Abcam; phalloidin (A12379) from Life Technologies.

Techniques: Disruption, Transfection, Western Blot, Control, Knockdown, Labeling, Staining, Confocal Microscopy