Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Interaction of GCase variants with their lysosomal transporter LIMP‐2. A) Crystal structure of GCase (PDB: 5LVX). [ ⁴⁴ ] The domains and motifs are colored as follows: pink: antiparallel beta‐sheet (domain 1); grey: TIM barrel (domain 2) containing active site (green); blue: beta‐barrel (domain 3); [ ⁴⁵ ] orange: proposed LIMP‐2‐binding motif. [ ⁴² ] Single amino acids comprising the active site, as well as amino acids exchanged in common disease‐associated GCase variants E326K, N370S, and L444P are labeled and highlighted. B) GCase activity in cell lysates of HEK 293T cells overexpressing GCase variants (n = 3‐8; mock: 8; wt: 8; E326K: 5; N370S: 3; L444P: 5; individual transfections). All disease‐associated variants show significantly reduced activity. E326K shows a residual activity of 40.0 ± 2.2%, N370S, and L444P do not differ significantly from the mock. C) Illustration of the lysosomal transport of GCase and its interaction with LIMP‐2. GCase binds to LIMP‐2 in the ER to form a transporting complex. After trafficking through the Golgi, the LIMP‐2/GCase complex is sorted to lysosomal compartments where low pH triggers complex dissociation. D) Western blot analyses of whole‐cell lysates and LE fractions of HEK 293T cells expressing GCase variants and FL‐LIMP‐2 or FL‐LIMP‐2‐3xD as a control. LE fractions are higher in GCase, LIMP‐2, and the lysosomal protein LAMP‐2A. Enriched fractions of cells overexpressing FL‐LIMP‐2 show increased levels of GCase. Blots containing a dashed line were spliced for a more comprehensive data presentation (both parts are from the same image). E) Quantification of western blot signal from lysate and LE HEK 293T samples (Figure ) (n = 3‐6; mock: 6; wt: 6; E326K: 3; N370S: 3; L444P: 3; individual cell harvests). The abundance of wt, E326K, and N370S GCase was significantly increased in LE fractions when FL‐LIMP‐2 was co‐expressed compared to the co‐expression of the non‐binding 3xD control. In general, the GCase signal was increased in LE fractions but did not reach statistical significance for FL‐LIMP‐2‐3xD samples and FL‐LIMP‐2 + L444P. F) GCase activity in cell lysates and LE fractions of HEK 293T cells overexpressing GCase variants and FL‐LIMP‐2 or FL‐LIMP‐2‐3xD (n = 3‐7; mock: 7; wt: 7; E326K: 4; N370S: 3; L444P: 4; individual cell harvests). Overexpression of FL‐LIMP‐2 resulted in an increase of GCase activity in the lysate itself, but also in the LE fraction for wt GCase and the E326K variant, while no significant differences were observed for the N370S and L444P variant. Asterisks (*) indicate significant differences between cell lysate and enriched fractions. Pound signs (#) indicate significant differences in relation to the respective 3xD control sample (example: wt + FL‐LIMP‐2 lysate versus wt + FL‐LIMP‐2‐3xD lysate). Statistics: replicates (dots) with a mean (column) ± SEM (B,E,F). Tests: One‐way ANOVA with Tukey's multiple comparison test (B); Two‐way ANOVA with Tukey's multiple comparison test (E,F). * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001; ## p < 0.01, #### p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Binding Assay, Labeling, Activity Assay, Transfection, Western Blot, Expressing, Control, Over Expression, Variant Assay, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Characterization of primary human fibroblasts of controls and patients with PD and GD. A) Western blot of whole cell lysates from control (CTRL‐1,2,3), PD patient‐derived (PD‐1,2), and GD patient‐derived (GD) primary human fibroblasts. Quantitative analysis of signals can be found in Figure (Supporting Information). GCase levels were comparable between control and PD but diminished in GD (Figure , Supporting Information). LIMP‐2 levels were increased in PD compared to controls (Figure , Supporting Information). LAMP‐2A and calnexin levels varied between cell lines and did not show any significant trend between groups (Figure , Supporting Information). GAPDH and CBB staining of the gel are presented as loading controls. B) GCase activity in whole‐cell lysates of control, PD, and GD fibroblast cell lines (n = 3‐7; CTRL‐1,2,3: 7; PD‐1,2: 7; GD: 3; individual cell harvests). The E326K lines PD‐1 and PD‐2 showed significantly lower GCase activity (66.41±2.69% and 73.56±6.50%) compared to the control lines. In contrast, activity in the GD line (GBA1 L444P/L444P ) was almost fully abolished (3.11±0.11% residual activity). C) Colocalization of LIMP‐2 and GCase in primary human fibroblasts determined via Pearson's correlation coefficient (n = CTRL‐1: 30; CTRL‐2: 38; CTRL‐3: 33; PD‐1: 44; PD‐2: 51; GD: 27; individual cells from multiple images). PD patient fibroblasts PD‐1 and PD‐2 show mildly reduced colocalization of LIMP‐2 and GCase compared to control lines. In the GD patient line, colocalization was diminished even further. D) Representative immunofluorescence images of primary human fibroblast lines. Objective magnification: 40x. Green: LIMP‐2; red: GCase, blue: DAPI. All lines show the vesicular distribution of LIMP‐2, indicating lysosomes. Control and PD lines show visible colocalization of GCase with the LIMP‐2 signal. In GD fibroblasts, the GCase signal was less intense and less granular, representing lower expression and lysosomal localization. Statistics: replicates (dots) with mean (column) ± SEM (B); violin plot with median (dashed line) and quartiles (dotted line) (C). Tests: Nested one‐way ANOVA with Tukey's multiple comparison test (B,C). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Western Blot, Control, Derivative Assay, Staining, Activity Assay, Immunofluorescence, Expressing, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Purification of GCase utilizing soluble (s) LIMP‐2. A) Workflow for the purification of sLIMP‐2/GCase complex from HEK 293F cells. A culture is transfected to co‐express sLIMP‐2 and GCase. After 96 h, the conditioned medium is collected and Ni‐NTA purification of His‐tagged proteins is performed, yielding sLIMP‐2 and sLIMP‐2/GCase complex. Elution fractions are concentrated and separated via SEC, yielding purified sLIMP‐2/GCase protein complex. B) Cartoon of intracellular mechanisms of interaction and secretion of GCase with the soluble sLIMP‐2 construct. A soluble sLIMP‐2/GCase complex is formed in the ER. The IgK leader sequence on sLIMP‐2 then facilitates secretion of the LIMP‐2/GCase complex instead of sorting GCase to the lysosome. In comparison, the sLIMP‐2‐3xD control construct does not bind GCase and therefore does not facilitate secretion of GCase. C) GCase activity in the supernatant of HEK 293F cells overexpressing wt GCase along either sLIMP‐2 or sLIMP‐2‐3xD (n = 3; individual transfections). Samples were taken 0‐72 h after transfection. In co‐expression with sLIMP‐2, GCase activity is higher and stable over the course of the measurements, whereas activity in the control is lower and diminishes over time. The statistical significance indicated (*) represents a comparison between both sample groups at a given time point. D) Representative western blot of analytical samples from Ni‐NTA purification of the sLIMP‐2/GCase complex from HEK 293F supernatant. Parts of the blot are shown with increased contrast to visualize faint signals. Splicing is indicated by a dashed line. Abbreviations: cond. SN: conditioned supernatant; dep. SN: depleted supernatant; wash 1/4: flow‐through of washing steps 1/4 (4 total); elution F1: elution fraction 1 (5 total). Elution of protein from Ni‐NTA resin yielded sLIMP‐2 and GCase. E) Representative SEC profiles of samples from Ni‐NTA purification of sLIMP‐2/GCase (blue), sLIMP‐2 (black), and His‐tagged GCase (grey, dashed) using a Superdex 200 Increase 3.2/300 column. sLIMP‐2 and sLIMP‐2/GCase samples share a peak at fraction 8 corresponding to the sLIMP‐2 monomer. An additional peak in fraction 7 is visible in the sLIMP‐2/GCase sample, corresponding to the sLIMP‐2/GCase complex. His‐tagged GCase runs as a major peak in fractions 4/5 with training smaller peaks in later fractions. F) Western Blot analyses of SEC fractions (corresponding to Figure ). Fractions of the sLIMP‐2/GCase sample show strong GCase and LIMP‐2 signals with maxima at fractions 7/8 and 8/9 respectively. Fractions of sLIMP‐2 show the same distribution with a weak GCase signal. His‐tagged GCase was most abundant in fractions 5/6. Statistics: replicates (dots, squares) with mean (line) ± SEM (C). Tests: Two‐way ANOVA with Sidak's multiple comparison test (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001 .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Purification, Transfection, Construct, Sequencing, Comparison, Control, Activity Assay, Expressing, Western Blot

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Design and effect of LIMP‐2‐derived helix 5 peptide on GCase function. A) Sequence of custom LIMP‐2 peptides. The peptides comprise the wt or 3xD variant of helix 5 of LIMP‐2 (orange), flanked by an N‐terminal lysosomal KFERQ sequence for lysosomal targeting (green) and a C‐terminal TAT‐peptide for cell penetration (pink). Linker regions are indicated in grey. B) Cartoon of uptake of LIMP‐2‐derived peptide into cells and the lysosome, where the peptide interacts with GCase, boosting its lysosomal function. C,D) Interaction of L2H5‐wt and GCase‐His at cytosolic (C) and lysosomal (D) pH as determined by MST (n = 3 sample preparations per condition). Dots represent mean ± SEM. With increasing concentration of ligand (L2H5‐wt), changes in the MST signal (FNorm) could be observed at both conditions, indicating binding to GCase. Fitting of a Kd model (red line) yielded estimated affinities in the nanomolar range for pH 7.4 and micromolar range for pH 5.0. At pH 7.4, higher L2H5 concentrations lead to a second change in the MST signal, hinting toward a second binding event with lower affinity (illustrated as a grey dashed line). Grey datapoints were disregarded for the Kd model fit (red line). E) Enzyme activity of Cerezyme in the presence of varying concentrations of L2H5‐wt or −3xD peptides (n = 3; individual experiments). An activating effect of L2H5‐wt was first observed in the micromolar range and increased further with peptide concentration. The addition of 10 µM of L2H5 led to a 2.63 ± 0.22‐fold increase of GCase activity. At peptide concentrations above 20 µM, precipitation of the peptide occurred as indicated by a dashed grey line. F) Effect of L2H5 peptides on the activity of recombinant GCase in conditioned HEK 293F media after overexpression of GCase variants (n = 3, individual experiments). The activity of wt GCase and E326K were increased in the presence of 10 µM L2H5‐wt. The activity of N370S and L444P were unaffected. Statistics: mean (dot) ± SEM (C,D), replicates (dots, squares) with mean (line) ± SEM (E); Mean (column) ± SEM (F). Tests: non‐linear regression Kd model (C,D); two‐way ANOVA with Tukey's multiple comparison test (F). * p < 0.05, **** p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Derivative Assay, Sequencing, Variant Assay, Concentration Assay, Binding Assay, Activity Assay, Recombinant, Over Expression, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Uptake and effect of L2H5 peptides on control and PD fibroblasts. A) Assessment of cell death via LDH activity in culture medium in CTRL‐2, PD‐1, and PD‐2 fibroblast lines (n = 3; wells from the 96‐well plate). Cells were treated with varying concentrations of L2H5‐wt peptide for 72 h. concentrations up to 10 µM did not lead to an increase in cell death. At a concentration of 20 µM however, LDH activity in the medium was significantly increased, indicating increased cell death due to treatment. Effects were comparable between all three lines. Significance is shown in comparison to the 0 µM data group for each cell line respectively. B) Presence of tryptic exogenous L2H5‐wt and endogenous LIMP‐2‐derived peptides in LE fractions of HEK293T cells after treatment with PBS (neg. ctrl.) or 5 µM L2H5‐wt for 2 h and 24 h. Determined via mass spectrometry. The positive control represents a sample spiked with 0.5 µg of L2H5 before analysis. L2H5‐wt‐specific peptides 1‐4 were detected in the pos. ctrl (all 4) and the cells treated with L2H5‐wt for 2 h (peptides 2 and 3). Low amounts of peptides 2 and 3 were also detected at the 24 h time point, but only by matching (indicated with an asterisk) and only in two of the three samples. Peptide 5, which is a tryptic product of both L2H5‐wt and endogenous LIMP‐2, was detected in all samples as expected, with higher abundance in the spiked control and the 2 h treated samples. C) Representative immunofluorescence image of CTRL‐2 fibroblasts with GFP‐labeled lysosomes (CellLight Lysosomes‐GFP) after 2 h of treatment with 0.25 µg µL −1 FRed‐L2H5‐wt. Objective magnification: 63x. Top: single channels. Middle: merged picture. Bottom: zoomed in single channels and merged picture of area inside a white frame. Green: GFP; red: FusionRed; blue: DAPI. FusionRed signal dots were visible within the cell. Some dots were surrounded by GFP signal located in the lysosomal membrane, thus confirming the presence of FRed‐L2H5‐wt inside the lysosome as shown by white arrows and in the zoomed‐in section. D) Live cell GCase activity in primary human control fibroblasts and PD‐patient‐derived fibroblasts harboring E326K mutations (n = 3; wells of a 96‐well plate). The cells were treated with PBS or L2H5 peptides (wt and 3xD). The graph shows lysosomal GCase activity as an area between curves (see materials and methods). In all three cell lines, lysosomal GCase activity was dramatically boosted after treatment with L2H5‐wt. In contrast, treatment with the non‐binding L2H5‐3xD peptide did not affect lysosomal GCase activity. See Figure (Supporting Information) for individual activity graphs with replicates. Statistics: replicates (dots) with mean (column) ± SEM (A); mean (column) ± SEM (D). Tests: Two‐way ANOVA with Dunnett's multiple comparison test (A); Two‐way ANOVA with Tukey's multiple comparison test (D). * p < 0.05, *** p < 0.001 **** p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Control, Activity Assay, Concentration Assay, Comparison, Derivative Assay, Mass Spectrometry, Positive Control, Immunofluorescence, Labeling, Membrane, Binding Assay

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: EV-A71 infection upregulated TRIB3 expression. (a) HCT-8 cells, RD cells and FHC cells were infected with EV-A71 (MOI = 1.0) and harvested at the indicated time post infection. The mRNA level of TRIB3 was analyzed by qRT-PCR ( n = 3). P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test. (b) EV-A71 infection increased TRIB3 expression in vitro in a time dependent manner. HCT-8 cells, RD cells and FHC cells were infected with EV-A71 (MOI = 1.0) and harvested at the indicated time post infection. Cell extracts were applied for WB assay. (c) EV-A71 infection increased TRIB3 expression in vitro in an inoculum dose dependent manner. HCT-8 cells, RD cells and FHC cells were infected with EV-A71 at the indicated MOI (MOI = 0.01, 0.1, 1.0) and harvested at 10 h post infection. Cell extracts were applied for WB assay.

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Infection, Expressing, Quantitative RT-PCR, In Vitro

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 enhances EV-A71 replication. (a to e) TRIB3 overexpression promoted EV-A71 infection. HCT-8 cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h after transfection, HCT-8 cells were mock-infected or infected with EV-A71 (MOI = 0.01) for 24 h. The cells were harvested for WB assay with indicated antibodies (a, n = 3), In-cell WB assay with indicated antibodies (b), IF (c), qRT-PCR assay (d, n = 3) or virus titer assay (e, n = 3) 24 h post infection. (f to j) TRIB3 depletion reduced the replication of EV-A71. Parental or TRIB3-KO HCT-8 cells were infected with EV-A71 (MOI = 0.1) for 24 h. The cells were harvested for WB assay with indicated antibodies (f, n = 3), In-cell WB assay with indicated antibodies (g), IF (h), qRT-PCR assay (i, n = 3) or titer assay (j, n = 3) 24 h post infection. (k) TRIB3-KO cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h after transfection, TRIB3-KO cells were mock-infected or infected with EV-A71 (MOI = 0.1) for 24 h. The cells were harvested for WB assay with indicated antibodies. P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test (e, j) or Student’s t -test (a, d, f, i).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Over Expression, Infection, Transfection, Quantitative RT-PCR, Virus, Titer Assay

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 does not interact with viral proteins. (a) 293 T cells were co-transfected with a plasmid expressing Myc-tagged TRIB3 and plasmid expressing HA-tagged VP1, 2A, 2B, 2C, 3A, 3C or GFP-tagged VP2-4, 3B, 3D protein or V5-tagged 2A. Immunoprecipitation from cell lysates were performed and probed with the indicated antibodies. (b) 293 T cells were transfected with plasmid expressing TRIB3-Myc or NEDD8-Myc, the cells were infected with EV-A71 at 24 h post transfection and harvested at 24 h post infection. Immunoprecipitation from cell lysates were performed and probed with the indicated antibodies.

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Infection

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 enhances SCARB2 expression and EV-A71 attachment to cells. (a) TRIB3 depletion decreased the protein level of SCARB2 . Tandem Mass Tag™-LC-MS/MS analysis evaluated the protein level of SCARB2 in WT cells and TRIB3-KO cells ( n = 2). (b, c, g, i) TRIB3 depletion decreased the protein level of SCARB2. SCARB2 expression were detected with WB (b, n = 3), qRT-PCR assay (c, n = 4), Immunofluorescence (g) and flow cytometry (i, n = 3) in WT cells and TRIB3-KO cells. (d, e, f, h) TRIB3 overexpression increased the protein level of SCARB2. HCT-8 cells were treated with Control-HA or TRIB3-HA plasmids. At 24 h after transfection, cells were harvested for WB assay with indicated antibodies (d, n = 3), qRT-PCR assay (e, n = 4), Immunofluorescence (f) and flow cytometry (h, n = 3). Percentage of SCARB2 positive cells was calculated with FCS express software. (j, k) TRIB3 overexpression promoted EV-A71 binding and TRIB3 knockout decreased EV-A71 binding. HCT-8 cells were transfected with indicated plasmids. At 24 h after transfection, HCT-8 cells were rested at 4°C for 1 h and infected with EV-A71 (MOI = 1.0) on ice for 30 min. The cells were rinsed with PBS and harvested. Cell-associated EV-A71 RNA were measured by a qRT-PCR assay (j. n = 3). WT cells and TRIB3-KO cells were rested at 4°C for 1 h and then infected with EV-A71 (MOI = 1.0) on ice for 30 min. The cells were rinsed with PBS and harvested. Cell-associated EV-A71 RNA were measured by were a qRT-PCR assay (k, n = 3). P < 0.05 , Student’s t -test (b, c, d, e, h, i, j, k).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR, Immunofluorescence, Flow Cytometry, Over Expression, Transfection, Software, Binding Assay, Knock-Out, Infection

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 represses ubiquitylation and degradation of SCARB2. (a) 293 T cells were co-transfected with a plasmid expressing SCARB2-Myc and TRIB3-HA or a vector plasmid. At 24 h post transfection, cells were incubated with cycloheximide (CHX) (10 μg/ml) for indicated times. Proteins were detected by WB with the indicated antibodies. P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test. (b) The effect of TRIB3 overexpression on SCARB2 ubiquitylation in vitro . 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-Myc Ab. The ubiquitylated SCARB2 was detected with WB assay. (c, d) 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-Myc Ab or anti-Flag Ab. The ubiquitylated SCARB2 was detected with WB assay. (e) The KDC region of TRIB3 was responsible for its promoting EV-A71 infection. (Upper panel) Schematic diagram of TRIB3 deletion mutants. (Lower panel) Cell extracts from 293 T cells transfected with the indicated plasmids and infected with EV-A71 were resolved by SDS-PAGE, proteins were detected by WB with the indicated antibodies. (f) Cell extracts from TRIB3-KO cells transfected with the indicated plasmids and infected with EV-A71 were resolved by SDS-PAGE, proteins were detected by WB with the indicated antibodies. (g) Effect of KDC deletion in TRIB3 on SCARB2 ubiquitination in 293 T cells. (h) Cell extracts from 293 T cells transfected with the indicated plasmids and infected with EV-A71 were detected by WB with the indicated antibodies. (i) The relationship between TRIB3 and SCARB2. 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-HA or anti-Flag Ab. (j) The relationship between endogenous TRIB3 and SCARB2. HCT-8 cells extracts were IP with anti-TRIB3.

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Over Expression, In Vitro, Infection, SDS Page

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 facilitates EV-A71 replication in a SCARB2-independent manner. (a) HCT-8 cells were transfected with control-HA or TRIB3-HA plasmids. At 3 h or 10 h post transfection, HCT-8 cells were harvested for WB assay with indicated antibodies. (b) HCT-8 cells were transfected with control-HA or TRIB3-HA plasmids. At 3 h post transfection, HCT-8 cells were mock-infected or infected with EV-A71 (MOI = 1.0) for 7 h. The cells were harvested for WB assay with indicated antibodies. (c) 293 T cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h post transfection, the cells were transfected again with EV-A71 subgenomic replicon RNA and luciferase reporter activities were determined at different time ( n = 6). (d) HCCLM3 cells and SCARB2-KO HCCLM3 cells were harvested for WB assay with indicated antibodies. (e) SCARB2-KO HCCLM3 cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h post transfection, the cells were transfected again with EV-A71 subgenomic replicon RNA and luciferase reporter activities were determined at different time ( n = 6). (f–g) SCARB2-KO HCCLM3 cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h after transfection, cells were mock-infected or infected with EV-A71 (MOI = 1) for 24 h. The cells were harvested for WB assay with indicated antibodies (f, n = 3) and qRT-PCR assay (g, n = 3). P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test (c, e) or Student’s t -test (f, g).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Transfection, Infection, Luciferase, Quantitative RT-PCR

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: Trib3 knockdown significantly reduce the lethality and severity of EV-A71 infection in mice. (a) Schematic presentation of animal experiment design. (b, c) Trib3 knockdown could delay the death of mice upon lethal EV-A71 challenge and photos were taken at 5 dpi. (d) 12-day C57BL/6 WT mice and Trib3 −/+ mice were infected with 1 LD 50 of EV-A71, mice survival was observed every day until day 12. (e, f) 12-day C57BL/6 WT mice and Trib3 −/+ mice were infected with 0.1 LD 50 of EV-A71, mice were weighed daily (e) and observed for clinical scores for 13 days (f). P < 0.05, A Log-Rank (Mantel-Cox) test (c, d) or Ridit assay (f).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Infection

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: Impact of Trib3 knockdown on mortality rates and mean survival time (MST) of mice infected with lethal EV-A71.

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Infection

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: Trib3 knockdown significantly attenuated EV-A71 replication in mice. (a to d) 12-day C57BL/6 WT mice and Trib3 −/+ mice were infected with 1 LD 50 of EV-A71. Three mice enrolled in each group were dissected at 3 or 5 dpi. Muscle tissues proteins were detected by WB assay with indicated antibodies (a) and viral titer assays (b). Paraffin-embedded sections of muscle tissues were prepared from mice at 3 or 5 dpi and examined with H&E stain (c). The muscle tissue sections prepared from mice at 3 or 5 dpi were stained with EV-A71 VP1 antibody for IHC analyses (d). P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test (a, b).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Infection, Staining

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Schematic diagram illustrating the workflow for human CRISPR/Cas9 library screening. b The normalized read counts of all sgRNAs in CRISPR/Cas9 library plasmid ( n = 29,957) and HCCLM3 cells infected with human CRISPR/Cas9 library ( n = 29,875). The center line indicates the 50th percentiles ; bounds of box = 25th–75th percentiles ; whiskers = 10th–90th percentiles; minima: bottom whiskers; maxima: top whiskers. c Representative images of tumorspheres. large spheres (>70 µm); smaller spheres (<40 µm). d MAGeCK analysis and RRA ranking of top enriched genes in small tumorspheres compared to large tumorspheres. e Ranked dot plots of top enriched genes in small tumorspheres compared to large tumorspheres. P value was obtained by permutation test using Benjamini-Hochberg procedure by MAGeCK. f The normalized read counts of sgRNAs targeting SCARB2 between large tumorspheres and small tumorspheres. g Flow cytometric analysis of the proportion of CD24, EpCAM, CD13, or CD133 positive cells in CTRL Cas9 or SCARB2 Cas9 HCCLM3 cells. h Real-time PCR analysis of SCARB2 expression in CD133 + CD13 + and CD133 - CD13 - HCCLM3, HepG2 and primary HCC cells. i Cell growth curves of CD133 + CD13 + and CD133 - CD13 - HCCLM3 and HepG2 cells with or without SCARB2 deletion. j Representative images of tumorspheres of indicated cells with or without SCARB2 knockout. The number of spheres was counted. Scale bar, 50 μm. k Effect of SCARB2 depletion on sorafenib sensitivity in HCCLM3 and HepG2 cells. The data are a summary of IC 50 values for sorafenib. l Representative immunofluorescence images and quantification of the viability of HCC organoids with indicated treatment. Calcein acetoxymethyl (calcein-AM) was used to mark viable cells (green) and ethidium bromide homodimer-1 to mark dead cells (red) ( n = 10 organoids per group). Scale bar, 50 μm. m Relative cell viabilities of tumor organoids with or without SCARB2 knockout . n Representative images and quantification of protrusive invasion of HCC organoids. Scale bar, 50 μm. o Flow cytometric analysis of the proportion of CD133, CD13, EpCAM or CD24 positive cells in HCC organoids with or without SCARB2 knockout. p IHC staining of SCARB2 expression in human normal liver tissues and HCC specimens ( n = 90 per group). q Kaplan–Meier survival curves for patients with HCC stratified by SCARB2 expression. ( g , h , i , j , k , m , n , o ) n = 3 biological repeats. Statistical significance was calculated by ( g , h , i , j , l , m , n , o , p ) two-tailed Student’s t test; ( q ) two-sided log-rank test; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: CRISPR, Library Screening, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Expressing, Knock-Out, Immunofluorescence, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Scheme used to establish the model of spontaneous HCC with targeted Myc knock-in and Scarb2 knockout in the liver. b Representative photographs (top) and H&E staining (bottom) of intrahepatic tumor tissues in the indicated mice 8 weeks after birth. Scale bar, 1 cm. H&E staining Scale bar, 100 μm. c The liver weights of WT ( n = 12 mice), Cre Alb My c ( n = 12 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 6 mice). d Incidence of HCC in Cre Alb My c ( n = 11 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 10 mice). e Kaplan–Meier survival curves for Cre Alb My c ( n = 11 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 10 mice). f The tumor initiation efficiency of HCC cells harvested from indicated group was evaluated by in vivo limiting dilution assay ( n = 10 mice per group). g Flow cytometric analysis of the proportion of EpCAM, CD133 or CD24 positive cells in indicated group ( n = 3 mice per group). h Scheme used to establish DEN-induced HCC mouse model. i Representative photographs (top) and H&E staining (bottom) of intrahepatic tumor tissue in the indicated mice 8 months after birth. j Liver weights of the Cre Alb mice ( n = 8 mice) and Cre Alb Scarb2 F/F mice ( n = 8 mice) in DEN-induced HCC mouse model. k Numbers of tumor nodules in the indicated mice ( n = 8 mice per group). l Effect of SCARB2 knockout in HCCLM3 cells on tumor growth ( n = 8 mice per group). Representative images of tumors ( m ) and tumor weights ( n ) in the indicated group ( n = 8 mice per group). o–q Effects of SCARB2 knockout in HCCLM3 spheroids on tumor growth, tumor sizes and tumor weights ( n = 8 mice per group). r Flow cytometric analysis of the proportion of CD24, EpCAM, CD13, or CD133 positive cells in tumors generated from HCCLM3 spheroids with or without SCARB2 knockout ( n = 8 mice per group). Statistical significance was calculated by ( c , g , j , k , l , n , o , q , r ) two tailed Student’s t test; ( d, e ) two-sided log-rank test; ( f ) one-sided extreme limiting dilution analysis. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Knock-In, Knock-Out, Staining, In Vivo, Limiting Dilution Assay, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Approaches for RNA-seq of HCC cells in the indicated groups. b The top Hallmark gene sets enriched in HCC cells from Cre Alb Myc mice compared to Cre Alb Scarb2 F/F Myc mice. P value was determined by one-sided permutation test. Statistical adjustments were made for multiple comparisons. c , d GSEA showing the enrichment of MYC target genes in Cre Alb Scarb2 F/F Myc vs Cre Alb Myc groups. P value was determined by one-sided permutation test. Statistical adjustments were made for multiple comparisons. e Visualization of SCARB2 positive and negative cells in tumorspheres by UMAP. f The proportion of SCARB2 positive cells in tumorspheres was analyzed by scRNA-seq. g , h Visualization of SCARB2 and MYC target genes expression in tumorspheres by UMAP. i Violin plot showing expression levels of MYC target gene score in SCARB2 positive and negative cells. The tips of the violin plot represent minima and maxima, and the width of violin plot shows the frequency distribution of data. j Correlation expression of MYC target genes and SCARB2 expression in SCARB2 positive cells. Each data point represents the value from an individual cell. k Effects of SCARB2 deletion or overexpression on the transcriptional activity of MYC. l Heatmap showing occupancy of genome-wide MYC peaks in CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells in a ± 3 kb window surrounding the TSS. m Metagene plots of global MYC occupancy in gene bodies in CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells. n CUT & tag tracks showing the binding of MYC to CDK4 and SLC2A1 in CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells. o , p CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells ( o ) or HCC cells from Cre Alb Myc and Cre Alb Scarb2 F/F Myc mice ( p ) were analyzed by ChIP with MYC or IgG antibody. ChIP’d DNA was quantified using qPCR for MYC or IgG binding to MYC target genes promoters. q Effect of SCARB2 knockout on the interaction MYC with MAX. Extracts of CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells were IP anti-MYC Ab and blotted with an anti-MAX Ab. r Colocalization of MYC and MAX in CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells was detected by the Duolink PLA assay. Data are representative images of MYC/MAX foci (left) and quantification of the number of fluorescent foci ( n = 8 cells per group). Scale bar, 5 μm. k , o , p , q , r n = 3 biological repeats. Statistical significance was calculated by ( i , k , o , p , r ) two tailed Student’s t test; ( j ) two-sided Pearson’s correlation test; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: RNA Sequencing Assay, Expressing, Over Expression, Activity Assay, Genome Wide, Binding Assay, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a The effects of SCARB2 deletion on the acetylation of MYC were assessed by Co-IP. b The effects of SCARB2 overexpression on the acetylation of MYC were assessed by Co-IP. c Effects of SCARB2 on the acetylation of MYC K148R mutants. HEK 293 T cells were transfected with the indicated plasmids for 24 h. Cell extracts were IP with an anti-MYC Ab. Acetylated MYC was detected by immunoblotting. d Effect of SCARB2 overexpression on HDAC3-mediated deacetylation. HCCLM3 cells were transfected with the indicated plasmids for 24 h. Cell extracts were IP with an anti-MYC Ab. Acetylated MYC was detected by immunoblotting. e Sequencing verification of the codon replacement by CRISPR-Cas9 resulting in MYC K148R . f Effect of HDAC3 overexpression on acetylation of MYC in MYC WT and MYC K148R HCCLM3 cells. g Effects of the MYC K148R mutation on MYC transcriptional activity in HCCLM3 cells with or without SCARB2 depletion. Data are means ± S.E.M of 3 independent experiments. h MYC WT and MYC K148R HCCLM3 cells were analyzed by ChIP with MYC or IgG antibody. ChIP’d DNA was quantified using qPCR for MYC or IgG binding to MYC target genes promoters, CAD , CDK4 , LDHA , NCL , PKM2 , HES1 , or Chr6 (negative control). i Effects of the K148R mutation on the interaction of MYC with BRD4, KAT5, and GCN5. j Effect of the MYC K148R mutation on the proliferation of HCCLM3 cells with or without SCARB2 depletion . k Effects of the MYC K148R mutation on the sphere-forming ability of HCCLM3 cells with or without SCARB2 depletion. l Schematic showing the role of MYC K148 acetylation in providing a potential docking site for binding with GCN5, KAT5, and BRD4. a , b , c , d , f , g , h , i , j , k n = 3 biological repeats. Statistical significance was calculated by ( g , h , j , k ) two tailed Student’s t test. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Over Expression, Transfection, Western Blot, Sequencing, CRISPR, Mutagenesis, Activity Assay, Binding Assay, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: Summary of mass spectrometry (MS) analysis of acetylation site of MYC in SCARB2-overexpressing HCCLM3 cells

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Mass Spectrometry

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a MS analyzed the interaction proteins of SCARB2 in HCCLM3 cells. b The interaction between MYC and SCARB2 in HCCLM3 cells was evaluated by Co-IP assays. c CO-IP analysis of SCARB2 and MYC interaction in the cytoplasm, cytomembrance and nucleus of HCCLM3 cells. d Co-localization of MYC/SCARB2 was detected in HepG2 cells with immunostaining. Scale bar, 5 μm. e The interaction between MYC and HDAC3 in CTRL Cas9 and SCARB2 Cas9 HCCLM3 cells was evaluated by Co-IP assays. f Co-localization of MYC and HDAC3 was detected in CTRL Cas9 and SCARB2 Cas9 HepG2 cells by immunostaining. Scale bar, 20 μm. g Mapping of MYC regions binding to SCARB2 and HDAC3. Left: deletion mutants of MYC. Right: HEK 293 T cells were cotransfected with the indicated constructs of MYC (GFP tag) and SCARB2 (Myc tag) or HDAC3 (HA tag). Cell extracts were IP with an anti-Myc Ab or anti-HA Ab. h Mapping of SCARB2 regions binding to MYC. HEK 293 T cells were cotransfected with the indicated constructs of SCARB2 (Myc-tagged) and MYC (Flag-tagged). Cell extracts were IP with an anti-Flag. i HCCLM3 cells treated with or without HDAC3 inhibitor (RGFP966 5μM) were analyzed by ChIP with MYC or IgG antibody. ChIP’d DNA was quantified using qPCR for MYC or IgG binding to MYC target genes promoters, CAD , CDK4 , LDHA , NCL , PKM2 , HES1 , or Chr6 (negative control). j Relative cell viabilities of HCCLM3 SCARB2 cas9 cells or HepG2 SCARB2 cas9 cells with overexpression of the indicated genes for the indicated times. k Sphere-forming capacity of HCCLM3 SCARB2 cas9 cells with overexpression of HDAC3 or HDAC3 R265P mutation. b , c , d , e , f , g , h , i , j , k n = 3 biological repeats. Statistical significance was calculated by ( i , j , k ) two tailed Student’s t test. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Immunostaining, Binding Assay, Construct, Negative Control, Over Expression, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a – b Virtual screening of FDA-approved drugs to identify small molecules binding with SCARB2 ( a ), top 10 hits are listed ( b ). c The kinetics of the SCARB2-PMB interaction were determined by surface plasmon resonance (SPR) analysis. d The highest scoring docking model of the SCARB2 and PMB complex is shown. Top: surface of the PMB-SCARB2 complex. Bottom: 3D structure of the PMB (yellow)-SCARB2 complex. e The kinetics of the SCARB2-MYC interaction with or without PMB were determined by SPR. f Structured illumination microscopic (SIM) images of vehicle- or PMB- treated HCCLM3 cells (1 h) stained for MYC and SCARB2. Scale bar, 10 μm. g The effect of PMB on the interaction of MYC and SCARB2 was evaluated by Co-IP assays. Extracts of DMSO and PMB-treated HCCLM3 cells were IP with an anti-SCARB2 Ab. h Representative images of MYC/HDAC3 colocalization foci in HCCLM3 cells before and after PMB treatment. Scale bar, 10 μm. i Effect of PMB on MYC acetylation. Extracts of DMSO and PMB-treated HCCLM3 cells were IP with an anti-MYC Ab. Acetylated MYC was detected by immunoblotting. j HCCLM3 cells treated with or without PMB were analyzed by ChIP with MYC or IgG antibody. ChIP’d DNA was quantified using qPCR for MYC or IgG binding to MYC target genes promoters. k Representative images and quantification of tumorspheres formed by HCCLM3 cells with indicated treatment. l Representative images and quantification of the viability of human-derived HCC organoids in 3D culture following the indicated treatment (10 organoids per group). m The frequency of tumor-initiating cells of HCC cells with or without PMB treatment were detected by in vitro limiting-dilution assays ( n = 10 per group). n – o Effects of the MYC K148R mutation on the sphere-forming ability ( n ) and proliferation ( o ) of HCCLM3 cells with or without PMB treatment. p – q Relative cell viabilities ( p ) and tumorsphere formation ( q ) of MYC- or SCARB2 -depleted HCCLM3 cells with or without PMB treatment. f , g , h , i , j , k , n , o , p , q n = 3 biological repeats. Statistical significance was calculated by ( j , k , l , n , o , q ) two tailed Student’s t test; ( m ) one-sided extreme limiting dilution analysis. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, SPR Assay, Staining, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, In Vitro, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Representative images of tumors and effects of the indicated treatments on tumor growth in the HepG2, HCCLM3 and Hepa1-6 CDX mouse models ( n = 8 mice per group). b Effects of the indicated treatments on tumor weights in the indicated CDX models ( n = 8 mice per group). c The tumor formation efficiency of HCC cells harvested from HepG2 and HCCLM3 CDX-derived HCC tumors was evaluated by in vivo limiting dilution assay. ( n = 8 mice per group). d Strategy for establishing PDX models from HCC patients. e The expression of MYC and SCARB2 were examined by WB in adjacent and HCC tissues from HCC patient of PDX model. Data are representative images from three independent experiments. f Representative images of tumors in the indicated PDX models ( n = 8 mice per group). g Effects of the indicated treatments on tumor growth in the indicated PDX model ( n = 8 mice per group). h Effects of the indicated treatments on the tumor weights in the indicated PDX models ( n = 8 mice per group). i Flow cytometry analysis for ALDH activity using the ALDEFLUOR kit in PDX model with indicated treatments. j Schematic diagram illustrates that SCARB2 drives hepatic carcinoma initiation by supporting cancer stem cell traits and enhancing MYC transcriptional activity. e , i n = 3 biological repeats. Statistical significance was calculated by ( a , b , g , h , i ) two tailed Student’s t test; ( c ) one-sided extreme limiting dilution analysis. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Human MYC -Flag-tag (HG11346-CF), SCARB2 -Flag-tag (HG11063-CF), SCARB2 -Myc-tag (HG11063-CM), MAX -His-tag (HG12885-CH), HDAC3 -HA-tag (HG11511-CY), and SIRT1 -HA-tag (HG10830-NY) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Derivative Assay, In Vivo, Limiting Dilution Assay, Expressing, Flow Cytometry, Activity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Purification of GCase utilizing soluble (s) LIMP‐2. A) Workflow for the purification of sLIMP‐2/GCase complex from HEK 293F cells. A culture is transfected to co‐express sLIMP‐2 and GCase. After 96 h, the conditioned medium is collected and Ni‐NTA purification of His‐tagged proteins is performed, yielding sLIMP‐2 and sLIMP‐2/GCase complex. Elution fractions are concentrated and separated via SEC, yielding purified sLIMP‐2/GCase protein complex. B) Cartoon of intracellular mechanisms of interaction and secretion of GCase with the soluble sLIMP‐2 construct. A soluble sLIMP‐2/GCase complex is formed in the ER. The IgK leader sequence on sLIMP‐2 then facilitates secretion of the LIMP‐2/GCase complex instead of sorting GCase to the lysosome. In comparison, the sLIMP‐2‐3xD control construct does not bind GCase and therefore does not facilitate secretion of GCase. C) GCase activity in the supernatant of HEK 293F cells overexpressing wt GCase along either sLIMP‐2 or sLIMP‐2‐3xD (n = 3; individual transfections). Samples were taken 0‐72 h after transfection. In co‐expression with sLIMP‐2, GCase activity is higher and stable over the course of the measurements, whereas activity in the control is lower and diminishes over time. The statistical significance indicated (*) represents a comparison between both sample groups at a given time point. D) Representative western blot of analytical samples from Ni‐NTA purification of the sLIMP‐2/GCase complex from HEK 293F supernatant. Parts of the blot are shown with increased contrast to visualize faint signals. Splicing is indicated by a dashed line. Abbreviations: cond. SN: conditioned supernatant; dep. SN: depleted supernatant; wash 1/4: flow‐through of washing steps 1/4 (4 total); elution F1: elution fraction 1 (5 total). Elution of protein from Ni‐NTA resin yielded sLIMP‐2 and GCase. E) Representative SEC profiles of samples from Ni‐NTA purification of sLIMP‐2/GCase (blue), sLIMP‐2 (black), and His‐tagged GCase (grey, dashed) using a Superdex 200 Increase 3.2/300 column. sLIMP‐2 and sLIMP‐2/GCase samples share a peak at fraction 8 corresponding to the sLIMP‐2 monomer. An additional peak in fraction 7 is visible in the sLIMP‐2/GCase sample, corresponding to the sLIMP‐2/GCase complex. His‐tagged GCase runs as a major peak in fractions 4/5 with training smaller peaks in later fractions. F) Western Blot analyses of SEC fractions (corresponding to Figure ). Fractions of the sLIMP‐2/GCase sample show strong GCase and LIMP‐2 signals with maxima at fractions 7/8 and 8/9 respectively. Fractions of sLIMP‐2 show the same distribution with a weak GCase signal. His‐tagged GCase was most abundant in fractions 5/6. Statistics: replicates (dots, squares) with mean (line) ± SEM (C). Tests: Two‐way ANOVA with Sidak's multiple comparison test (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001 .

Article Snippet: These were sLIMP‐2‐FC (Sino Biological Inc., Peking, China, #11063‐H03H), imiglucerase/Cerezyme (Sanofi Genzyme, Cambridge, MA, United States).

Techniques: Purification, Transfection, Construct, Sequencing, Comparison, Control, Activity Assay, Expressing, Western Blot