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. 2018 Nov 9;8:16644. doi: 10.1038/s41598-018-34523-3

Figure 8.

Figure 8

Lysosome function alteration in MPS VII NPCs and neurons. Autophagy basal levels in control and MPS VII NPCs (A) and in control and MPS VII neurons (B). P62 and LC3-I/ II were analyzed by western blot of control and MPS VII NPC protein extracts, non-treated (−) and treated (+) with rβ-gluc. Data from one representative experiment of 4 independent experiments; Images from different parts of the same gel. (C) Control and MPS VII NPC lysosome ability to degrade membrane proteins internalized by endosomes assayed by an EGFR degradation assay. EGFR content analyzed by western blot. Data from one representative experiment of 2 independent experiments; Image from different parts of the same gel. (D) Quantification of EGFR degradation efficiency in control and MPS VII NPC normalized to β-tubulin levels. (E) Immunofluorescence confocal microscopy of control and MPS VII NPC, after induction of CAR degradation, stained for LAMP1 (green), CAR (extra- and intracellular domains, red) and DAPI (blue). CAR localized at the membrane, internalized into lysosomes (LAMP1 and CAR co-localization, 30′) and degraded (120′). (F) Lysosome ability to degrade CAR assayed in control and MPS VII NPC, non-treated (non-treat.) and treated with rβ-gluc. CAR content analyzed by Western blot. Data from one representative experiment of 2 independent experiments; Control, MPS VII-8 and MPS VII-13 gels are independent and CAR and β-tubulin images are from different parts of the same gel. (G) Lysosome ability to degrade membrane proteins internalized by endosomes assayed by an EGFR degradation assay in control and MPS VII neurons, non-treated (N.T.) and treated with rβ-gluc. EGFR content analyzed by Western blot. Data from one representative experiment of 5 independent experiments; Image from different parts of the same gel. (H) Quantification of EGFR degradation efficiency in control and MPS VII neurons normalized to actin levels. (I) Control and MPS VII neurons lysosome ability to degrade CAR, non-treated (N.T.) and treated with rβ-gluc. CAR content was analyzed by Western blot. Data from one representative experiment of 5 independent experiments; Image from different parts of the same gel. (J) Quantification of CAR degradation efficiency in control and MPS VII neurons normalized to actin levels.