Fig 2: The effect of inhibition of macropinocytosis and dynamin-dependent endocytic trafficking on the expression of lysosomal proteins. (A) LO2 hepatocytes and HEK293 cells were transfected with pCMV6-CREG1 or the vector alone and cultured for 10 days. Cells were coimmunostained for CREG1 and IGF2R. (B) LO2 cells were immunoprecipitated (IP) with anti-CREG1 monoclonal antibody or pre-immune IgG. Immunoblotting was used to detect for IGF2R. (C) HEK293 cells stably transfected with pCMV6-CREG1 (T) or the vector alone (C) were immunoprecipitated with anti-IGF2R polyclonal antibody and immunoblots were probed for CREG1 and IGF2R. (D) Confluent LO2 cells were cultured in RPMI-1640 medium for 24 h. Conditioned media were immunoprecipitated with HEXA antibody or pre-immune IgG. Immunoblots were analyzed with the same antibody. Cell lysates were also analyzed by immunoblotting. (E) The percentage of HEXA in the conditioned media were plotted. N = 3, P = 0.785. (F) LO2 cells stably transfected with pCMV6-CREG1 or the empty vector were pre-incubated with the macropinocytosis inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA), dynasore (dyn, an inhibitor for dynamin-dependent endocytosis), or the vehicle control for 30 min. The cells were then labeled with TRITC-dextran (Dex) or Alexa Fluor 488-TF for 30 min. Treatment of the cells with EIPA and dynasore inhibited the internalization of TRITIC-Dex and Alexa Fluor 488-TF, respectively. (G) LO2 cells were treated with EIPA, dynasore, or the vehicle control (C) for 24 h and then harvested for immunoblot analysis. GAPDH served as a loading control. (H) The blots were quantified by densitometry and plotted as a ratio to GAPDH. N = 3, *P < 0.05 vs pCMV6-C, #P < 0.05 vs pCMV6-CREG1-C

Fig 3: Identification of the core promoter of human CREG gene (hCREG) and the transcription factors that bind to the hCREG promoter.(A) Identification of the core promoter of hCREG. A 1.9 kb genomic DNA sequence upstream of the hCREG transcription start site (TSS) linked with Xho I/Hind III restriction sites was subcloned into pGL4.12-basic luciferase reporter vectors. A series of promoter deletions were created based on the 1.9 kb hCREG-luciferase reporter. HUVECs and 293T cells were transiently transfected with reporter vectors and harvested after 48 h. For each construct, a PGL4.73 plasmid was co-transfected to correct for differences in transfection efficiency. The corrected luciferase activity was normalized to the activity of the hCREG -508/+78 plasmids (100% activity). (B) Promoter-binding transcription-factor (TF) profiling array assay of hCREG core promoter was performed. This is a competitive binding assay performed to identify promoter-bound TFs through comparisons of the results in the presence (control + promoter) or absence (control) of the hCREG core promoter. If the hCREG promoter contains a TF binding sequence, it will display a lower chemiluminescence activity. In this study, hCREG was found to potentially bind with Brn-3, C\EBP, EGR, ETS, GATA, GR/PR, HIF, IRF, NF-1, NF-?B and Pax-5. (C) Bioinformatic predictions of the consensus binding sequences of C\EBP ß, GATA1, and Ets-1. (D) ChIP assay confirmed that GATA1 bound directly to hCREG at the consensus GATA binding sequence [(-297/-292 bp) upstream from TSS]. (E) Plasmids containing the hCREG promoter (-508/+78) with 2 deletion mutations at the consensus GATA1 binding element were constructed with the luciferase (Luc) gene in PGL4.12 vectors. Luciferase activity was normalized to that of the hCREG core promoter (100%). n = 9. Data are shown as the mean ± SE from 3 independent experiments performed in triplicates. *P<0.05 and ***P<0.001 compared with either the hCREG core promoter (100%) (A and E) or the control (B).

Fig 4: CREG1 promotes lysosomal biogenesis. (A) LO2 cells stably transfected with pCMV6-CREG1 and control cells were analyzed by immunoblotting for lysosomal proteins and markers for the endoplasmic reticulum and the Golgi apparatus. ACTB served as a loading control. (B) Lysosomal mRNA levels in LO2 cells stably transfected with pCMV6-CREG1 or pCMV6 were analyzed by RT-PCR. RNA18S rRNA served as a loading control. (C) LO2 cells were incubated with 50 nM LysoTracker for 10 min, washed with PBS, and photographed by fluorescence microscopy. (D) Fluorescence intensity was quantified and plotted. N = 30 for each group. *P < 0.001. (E) AML12 cells with stable CREG1 knockdown (KD) and control cells were subjected to immunoblot analysis. ACTB served as a loading control. (F) Lysosomal gene transcripts in CREG1 knockdown and control AML12 cells were analyzed by RT-PCR. Rn18s rRNA served as a loading control. (G) CREG1 knockdown and control AML12 cells were incubated with LysoTracker for 10 min, washed with PBS, and photographed by fluorescence microscopy. (H) Fluorescence intensity was quantified and plotted. N = 30 for each group. *P < 0.001. (I) Immunoblots show absence of CREG1 in the liver of creg1-/- mice. ACTB served as a loading control. (J) Liver tissues of Creg1+/+ and creg1-/- mice (lanes 1 and 2, 3 and 4, and 5 and 6 were samples from littermates) were analyzed by immunoblotting for lysosomal proteins. GAPDH served as a loading control

Fig 5: CREG1 promotes endocytic trafficking. (A) LO2 cells stably transfected with human CREG1 cDNA (pCMV6-CREG1) or the vector alone were incubated with 100 µg/ml TRITIC-dextran for 30 min. The cells were then fixed with 3% paraformaldehyde and internalized TRITIC-dextran was visualized by fluorescence microscopy. (B) Fluorescence intensity of internalized TRITIC-dextran was quantified using ImageJ software. N = 30 for each group, *P < 0.001. (C) LO2 cells stably transfected with pCMV6-CREG1 or the empty vector were incubated with 50 µg/ml Alexa Fluor 488-TF (transferrin) for 30 min. Endocytosis of Alexa Fluor 488-TF was visualized by fluorescence microscopy. (D) Fluorescence intensity of internalized Alexa Fluor 488-TF was quantified and plotted. N = 30 for each group, *P < 0.001. (E) Mixed populations of CREG1-transfected and untransfected LO2 cells were incubated with 50 µg/ml Alexa Fluor 488-TF for 30 min. Cells were fixed and stained for CREG1. Arrows indicate colocalization of CREG1 with internalized Alexa Fluor 488-TF in the transfected cells. (F) LO2 cells were incubated with 50 µg/ml Alexa Fluor 488-TF at 4°C for 15 min, washed, and chased at 37°C for 30 or 60 min. The cells were then fixed and immunostained for RAB5. Arrowheads point to the accumulation of Alexa Fluor 488-TF at a subplasma membrane region. Arrows indicate colocalization of Alexa Fluor 488-TF with RAB5. (G) AML12 mouse hepatocytes were stably transfected with control or Creg1 shRNA (CREG1 knockdown, CREG1 KD). The cells were analyzed by immunoblotting for CREG1. ACTB served as a loading control. (H) CREG1 knockdown and control cells were incubated with 200 µg/ml FITC-ALB (albumin), fixed, and photographed by fluorescence microscopy. (I) Fluorescence intensity of internalized FITC-ALB was quantified. N = 30 for each group, *P < 0.001. (J) AML12 cells were incubated with 50 µg/ml Alexa Fluor 488-TF for 30 min and photographed by fluorescence microscopy. (K) Internalized Alexa Fluor 488-TF was quantified and plotted. N = 30 for each group, *P < 0.001