Fig 2: BFA-induced ER stress increases transcription and N-terminal cleavage of Luman.(a) Ethidium bromide stained agarose gel showing OASIS, BBF2H7, CREBH, AIbZIP, LUMAN, PRNP, HSPA5 and HPRT1 RT-PCR amplification products from MCF-7 cells (n = 4), neurons (n = 7) and astrocytes (n = 5) treated with DMSO (D, 0.1%), or 5 µg/mL of brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. The fold increase relative to DMSO of band intensity normalized to HPRT1 is indicated. (b,c) Relative increase in LUMAN (b) or HSPA5 (c) mRNA levels assessed by qPCR 18 h after DMSO, BFA, Th or TM treatments (5 µg/mL). Data represent the mean ± SEM of three experiments, analysed using a one-way ANOVA followed by a Dunnett post-hoc test *p < 0.05 compared to DMSO. (d) Ethidium bromide stained agarose gel showing LUMAN and HPRT1 RT-PCR amplification products from MCF-7 cells treated with DMSO or BFA (5 µg/mL) for 18 h, in the presence of 1 µg/mL actinomycin D (Act. D) or 20 µg/mL cycloheximide (CHX)). (e) Western blot analysis of ?Luman, BiP, and ß-Actin protein levels from MCF-7 cells or primary human neuron and astrocytes treated with 0.1% DMSO or 5 µg/mL of BFA, Th or TM for 18 h. (f) Ethidium bromide stained agarose gel showing MAP2, GFAP and HPRT1 RT-PCR amplification products from MCF-7 cells, neurons and astrocytes60. (g) Western blot analysis of HA-tagged ?Luman, eGFP and ß-Actin protein levels from HEK293T transfected with an N-terminal HA-tagged full length Luman treated with 0.1% DMSO or 5 µg/mL of BFA, Th or TM for 18 h. (h) Time course assessment of Luman cleavage in HEK293T transfected with N-terminal HA-tagged full length Luman, and treated with BFA (5 µg/mL) or DMSO (Ctl, 0.1%). Vector designates HEK293T cells transfected with the pBud-eGFP vector. Full-length images of blots and gels are presented in Supplementary Information.

Fig 3: Luman contributes to BFA-induced PRNP expression.(a) Relative increase in PRNP mRNA levels assessed by qPCR 24 h after ?Luman transfection in MCF-7 cells. Figure represents the mean ± SEM of five experiments, analysed using a unilateral student t-test *p = 0.01. (b) Western blot for PrP, ?Luman and ß-Actin of MCF-7 cells transiently transfected with ?Luman. Relative PrP/ß-Actin ratio is indicated, and represents the mean of nine experiments. (c) Relative increase in PRNP mRNA levels assessed by qPCR in MCF-7 cells transfected with scrambled (siCtrl) or Luman-targeting (siLuman) siRNA, and treated with DMSO (D, 0.1%) or 5 µg/mL brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. Data represent the mean ± SEM of seven experiments, analysed using a two-way ANOVA followed by a Bonferroni post-hoc test *p < 0.0001 compared to siCtrl. (d) Relative increase in PRNP mRNA levels assessed by qPCR in primary human neurons transfected with non-targeting (white) or Luman-targeting (black) siRNA and treated with DMSO (D, 0.1%) or BFA (5 µg/mL), for 18 h. Represents the mean ± SEM of five experiments, analysed using a two-way ANOVA followed by a Bonferroni post-hoc test *p < 0.05 compared to DMSO. Lower panels LUMAN and HPRT1 amplicons obtained by RT-PCR. (e) Western blot for PrP, ?Luman and ß-Actin of MCF-7 cells transfected for 24 h with scrambled (siCtrl) or Luman-targeting (siLuman) siRNA and treated with DMSO (0.1%) or 5 µg/mL of BFA, Th or TM for 18 h. Mature, immature and unglycosylated PrP bands are indicated. Full-length images of blots and gels are presented in Supplementary Information.

Fig 4: Atorvastatin-induced neuritogenesis is independent of Luman activity.(a) Representative image of neuritogenesis in N2a and SK-N-SH neuroblastoma cells following atorvastatin (ATV, 20 µM) treatment for 24 h. (b,c) Western blot for PrP and ß-Actin of N2a (b) or SK-N-SH (c) cells treated with 20 µM atorvastatin (ATV) for increasing amounts of time (0–24 hrs) or drug concentrations (0–20 µM). (d) Quantification of neurite-bearing N2a cells following treatment with 0.1% DMSO or 20 µM ATV for 24 h. Data represent the average percentage of neurite-bearing cells (mean ± SEM, n = 10 images), analysed using a unilateral student t-test *p < 0.001. (e) Quantification of neurite-bearing N2a cells transfected with scramble (siCtrl) or Luman-targeting siRNA (siLuman), following 0.1% DMSO or 20 µM ATV for 24 h. Inset Luman and Hprt1 amplicons obtained by RT-PCR from siCtrl- or siLuman-transfected N2a cells. Data represent the average percentage of neurite-bearing cells (mean ± SEM, n = 10 images), analysed using a two-way ANOVA. (f) Quantification of mean neurite length of N2a cells transfected with scramble (white) or Luman-targeting siRNA (black), following 20 µM ATV for 24 h. Data represent the average of total neuritic length (pixel)/total number of cells (mean ± SEM, n = 10 images), analysed using a bilateral student t-test. (g,h) Western blot for the Luman N-terminal HA tag, eGFP or ß-Actin of N2a (g) or HEK293T (h) cells transfected with pBud-eGFP (Vector) or an N-terminal HA-tagged full length Luman construct (pBud-eGFP-HALuman1-317), and treated with 0.1% DMSO (Ctl), 5 µg/mL brefeldin A (BFA) or 20 µM ATV, for increasing amounts of times (0–18 hrs). NS designates an unspecific band. Full-length images of blots are presented in Supplementary Information.