Fig 2: Gene and protein expression analysis of cathepsins B, D and L in iPSCs and neurons derived from CTRL and ULD patients. (Top panel) (A) qRT-PCR analysis of cathepsins mRNA expression in iPSCs. ULD patient-derived iPSCs show a higher expression of cathepsins compared to CTRL iPSCs. (B) Western blot analysis of total lysates obtained from CTRL and ULD iPSCs does not show an enhanced cathepsins expression in patients iPSCs compared to control. (C) Densitometric analysis of WB results, performed using ImageJ software. (Bottom panel) (D) qRT-PCR analysis of cathepsins mRNA expression in neurons at day 21 of differentiation. ULD patient-derived neurons show a higher expression of cathepsins genes compared to control. (E) Western blot analysis of total lysates obtained from CTRL and ULD neurons results in an overall increase of cathepsins protein expression (particularly cathepsins B and D for ULD2) in patients compared to control, (F) Densitometric analysis of WB results, performed using ImageJ software. In all WB analyses, GAPDH was used as loading control. qRT-PCR and WB data are each presented as mean ± SEM of three independent experiments (black dots), * p = 0.05, ** p = 0.01. For neurons, t-test has been calculated vs. CTRL at the same day of differentiation.

Fig 3: Family pedigree of ULD patients studied (created with Biorender). Open symbols: unaffected individuals, solid symbols: affected individuals, squares: males, circles: females, symbols with a diagonal line: deceased.

Fig 4: Quantification of the number of dodecamers by fragment length analysis. (Left) PCR amplification with 5'-FAM labelled primers of a portion of ~180 bp across the dodecamer repeats of the CSTB promoter, performed on genomic DNA extracted from CTRL and ULD iPSCs and visualization of the amplified fragments on agarose gel (100-bp DNA ladder was used as a size marker). The position of Fw and Rv primers (at -296 bp and -118 bp, respectively) is indicated. (Right) Fragment length analysis by capillary electrophoresis of PCR products. The sizes of the main fragments are indicated above the peaks in bp. At 180 bp is the peak for the CTRL (2 dodecamers); at 644 bp are the peaks for the first allele of ULD1 and ULD2 (41 dodecamers); at 921 bp is the peak for the second allele of ULD1 (64 dodecamers); at 909 bp is the peak for the second allele of ULD2 (63 dodecamers). GeneScan LIZ 1200 was used as marker size (orange peaks in CTRL electropherogram) and the data were analyzed by Eurofins with GeneMapper 6 software (Applied Biosystems). Representative images were created using Peak Scanner software (v. 2.0; Applied Biosystems).

Fig 5: Protein expression analysis of PARP1 in neurons derived from CTRL and ULD patients. (A) Western blot analysis of total lysates obtained from CTRL and ULD neurons results in high level of expression of full length PARP1 in patients compared to control; the expression of 89-kDa fragment produced by the cleavage of PARP1 is only detectable in patient’s cells. (B) Densitometric analysis of WB results, performed using ImageJ software. GAPDH was used as loading control. Data are presented as mean ± SEM of three independent experiments (black dots), ** p = 0.01, *** p = 0.001. t-test has been calculated vs. CTRL neurons at the same day of differentiation.