Journal: International Journal of Cancer

Article Title: Antitumor activity of an anti‐ CD 98 antibody

doi: 10.1002/ijc.29415

Figure Lengend Snippet: Antitumor activity of anti‐CD98 antibodies in Burkitt's lymphoma model ramos, biophysical properties of 18‐2A and IGN523 and comparison of their antitumor efficacy in ramos and dau Burkitt's lymphoma xenografts. ( a ) of note, in studies 1 and 2, rituximab was used as a positive control and elicited a TGI of 88% ( p < 0.001) and 68% ( p < 0.001), respectively. ( b ) biacore analysis of 18‐2A (murine IgG 2a ) and IGN523 (human IgG1) on recombinant human CD98 protein. ( c ) ramos or (d) dau growth curves in mice receiving 18‐2A (15 mg/kg), IGN523 (15 mg/kg) or control antibody (control IgG; 15 mg/kg) once weekly intraperitoneally (i.p.) ( n = 8); ( c ) *** p < 0.001 versus control, ( d ) * p < 0.05; ** p < 0.01 relative to control. Treatment started on day of randomization. Starting tumor volumes were 248 ± 112 and 111 ± 38 mm 3 for ramos and dau, respectively. Please note no statistical difference between 18‐2A and IGN523 treatment groups in ramos. Arrow = administration of antibody; N.S. = statistically not significant; n.a. = not applicable.

Article Snippet: Recombinant His‐tagged human CD98 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Activity Assay, Positive Control, Recombinant

Journal: International Journal of Cancer

Article Title: Antitumor activity of an anti‐ CD 98 antibody

doi: 10.1002/ijc.29415

Figure Lengend Snippet: Tumor growth inhibition of IGN523 in different tumor models. ( a ) HL‐60, ( b ) OCI‐AML‐3, ( c ) KG‐1, ( d ) IGN‐LNG‐12 and ( e ) IGN‐LNG‐54 growth curves in either NOD/SCID‐ ( c – e ) or CB.17‐SCID‐mice ( a and b ) receiving IGN523 (15 mg/kg) or control IgG (15 mg/kg). Carboplatin was administered in IGN‐LNG‐12 and IGN‐LNG‐54 at 10 mg/kg i.p. ( n = 8). ( f ) anti‐CD98 staining of representative IGN‐LNG‐54 tumors post‐treatment. * p < 0.05; *** p < 0.001 relative to control. Starting tumor volumes were 134 ± 32 mm 3 (HL‐60); 174 ± 34 mm 3 (OCI‐AML‐3); 92 ± 11 mm 3 (KG‐1); 126 ± 23 mm 3 (IGN‐LNG‐12) and 136 ± 38 mm 3 (IGN‐LNG‐54). Please note a statistical difference between IGN523 and carboplatin treatment groups in IGN‐LNG‐54 ( p < 0.04). Arrow = administration of antibody. Scale bar = 20 μm.

Article Snippet: Recombinant His‐tagged human CD98 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Inhibition, Staining

Journal: International Journal of Cancer

Article Title: Antitumor activity of an anti‐ CD 98 antibody

doi: 10.1002/ijc.29415

Figure Lengend Snippet: IGN523 demonstrates strong ADCC‐, but not CDC‐activity. Antibody‐induced luciferase activity in the effector cell is quantified with luminescence readout and is a surrogate for effector cell‐mediated cell lysis. Target cells ( a and d ) ramos, ( b and e ) KG‐1 and ( c ) OCI‐AML‐3 were used in ( a – c ) ADCC or ( d and e ) CDC assays. Please note that IGN523 elicits greater ADCC activity than rituximab in ramos cells. Cetuximab (anti‐EGFR) was used as negative control antibody (control IgG). Ramos, KG‐1 and OCI‐AML‐3 cell lines both endogenously express CD98 on the cell surface.

Article Snippet: Recombinant His‐tagged human CD98 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Activity Assay, Luciferase, Lysis, Negative Control

Journal: International Journal of Cancer

Article Title: Antitumor activity of an anti‐ CD 98 antibody

doi: 10.1002/ijc.29415

Figure Lengend Snippet: IGN523‐CD98 complex colocalizes with LAMP‐1. IGN523 induces lysosomal permeability. ( a ) ramos cells were incubated at 4 °C for 30 min with 2 μg/mL Alexa488‐labeled anti‐human CD98 antibody (green), 10 μg/mL anti‐human crosslinker (AbXL) and hoechst (blue). Cells were subsequently incubated at 37 °C for the indicated time points (0 or 90 min). LAMP1 was stained with anti‐LAMP1‐AF647 antibody (red). Note that CD98 and LAMP‐1 colocalize (yellow). Top row: 0 min; bottom row: 90 min; scale bar = 10 μm. ( b ) lysosomal volume and ( c ) lysosomal permeability was assessed in IGN523‐crosslinked ramos cells at 4 and 12 hr, respectively. The ratio of IGN523 (10 μg/mL):AbXL (50μg/mL) was 1:5. Cetuximab was used as control antibody (control IgG). *** p < 0.001 relative to IGN523.

Article Snippet: Recombinant His‐tagged human CD98 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Permeability, Incubation, Labeling, Staining

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: ( A ) Levels of glutamine in MCF10A cells transfected with Vector Ctrl, ΔATG1 + 2 or Flag-hSPAR ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( B ) Co-immunofluorescence staining of P27KIP1 (green) and Lyso-Tracker (red) in MDA-MB-231 cells cultured with or without glutamine. Cells were permeabilized with digitonin to remove the soluble P27KIP1. Nuclei were stained with Hoechst (blue). The graphs display the fluorescence intensity (arbitrary units) of P27KIP1 and Lyso-Tracker over the distance from adjacent image (depicted by the arrows). Scale bar, 5 µm ( n = 3 independent biological samples). ( C ) Immunoblotting against SLC7A1, SLC7A5, Flag and GAPDH in extracts from MDA-MB-231 cells transfected with Vector Ctrl, ΔATG1 + 2, or Flag-hSPAR ( n = 3 independent biological samples). ( D ) Quantified relative levels of SLC7A1/GAPDH and SLC7A5/GAPDH from panel ( C ) ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( E ) Immunoblotting of whole-cell extracts (upper panel), cytoplasmic (lysosome components removed) and lysosomal extracts (lower panel) prepared from MDA-MB-231 cells cultured with or without arginine, leucine or glutamine against P27KIP1, GAPDH, LAMP2 (lysosomal marker) and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( F ) Changes of interaction between P27KIP1 and TRIM21, P27KIP1 and LAMTOR1 were detected by Co-IP and immunoblotting in the indicated fractions extracts from MDA-MB-231 cells after transfection with the indicated constructs. Left, immunoblotting of inputs. Right, immunoblotting using antibodies against P27KIP1, TRIM21 and LAMTOR1 following IP of P27KIP1 ( n = 3 independent biological samples).

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Cell Culture, Fluorescence, Western Blot, Marker, Co-Immunoprecipitation Assay, Construct

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) has beneficial effects on cartilage maintenance in mice with osteoarthritis (OA). a) Schematic representation of the timeline of the antibody drug or recombinant protein (EDIL3 or CD98) treatment during 16 to 21 weeks of age. STR/ort mice were injected weekly through the tail vein with phosphate-buffered saline (PBS, vehicle) combined with either the antibody or the protein for six consecutive weeks. Mice were killed at 22 weeks. This was followed by paraffin tissue sections and Safranin O and immunofluorescence (IF) staining in knee articular cartilage. b) Representative images include articular cartilage in the tibial plateau knee joints. Arrows indicate representative matrix-producing (red) and matrix-non-producing (black) chondrocytes. c) The total chondrocyte number in the articular cartilage was quantified. EDIL3 protein treatment increased the number of chondrocytes. d) The number and percentage of matrix-non-producing chondrocytes (MNCs) in the articular cartilage were quantified. EDIL3 antibody treatment increased the number of MNCs. e) EDIL3 protein treatments decreased the Osteoarthritis Research Society International (OARSI) score in the LFC. f) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the EDIL3; 4',6-diamidino-2-phenylindole (DAPI) (blue) stained nuclei; orange dashed lines define the cartilage region. EDIL3 antibody treatments decreased the EDIL3 contents in the cartilage region. g) The fluorescence of EDIL3 was quantified in the whole articular cartilage region. EDIL3 antibody significantly decreased EDIL3 expression. h) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the indicated OA markers; DAPI (blue) stained nuclei; orange dashed lines define the cartilage region. i) Fluorescence was quantified in the whole articular cartilage region. Data are presented as means and standard errors in . Data are presented as mean and maximum with 95% confidence intervals in . CD98 protein significantly increased SOX9 expression, and EDIL3 protein significantly reduced aggrecan fragments. LTP, lateral tibial plateau; MFC, medial femoral condyle; MMP, matrix metalloproteinase; MTP, medial tibial plateau. Data presented in Figures 3c to 3e were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Data in Figures 3g to 3i were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Isotype Ab, immunoglobulin G (IgG)1 antibody.

Article Snippet: The STR/ort mice were administered tail-vein injections of 1 μg/mouse anti-EDIL3 IgG1 (MABS1976; MilliporeSigma), 2 μg/mouse recombinant EDIL3 protein (6046-ED; R&D Systems), 2 μg/mouse CD98 protein (50813-M07H; Sino Biological, China), 1 μg/mouse isotype-matched control IgG1 monoclonal antibody (mAb) (mabg1-ctrlm; InvivoGen, USA), or vehicle PBS solution.

Techniques: Recombinant, Injection, Saline, Immunofluorescence, Staining, Fluorescence, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) treatment prevents subchondral bone plate thickness (PI.Th) and epiphyseal trabecular mineralization. a) Representative cross and horizontal sections of knee joints obtained from STR/ort mice. The 3D reconstruction of a proximal tibia with a plane indicates the location from which the cross and horizontal sections were obtained. The vehicle control mice exhibited uneven trabecular bone distribution; moreover, EDIL3 antibody treatment was observed to worsen this phenomenon. However, EDIL3 and CD98 protein prevented subchondral bone mineralization. b) Trabecular bone morphometric parameters were calculated using micro-CT analysis. The medial and lateral subchondral bone plate and underlying epiphyseal trabecular bone were analyzed separately. Epiphysis was manually selected as representative of subchondral bone. The epiphyseal trabeculae were split from the subchondral bone plate, and trabecular bone morphometric parameters were calculated. EDIL3 protein treatment prevented osteoarthritis (OA)-associated reduction in subchondral bone total porosity (Po(tot)). EDIL3 protein treatment also decreased the subchondral PI.Th, bone volume (BV/TV), and trabecular parameters (trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp)). Analyses were conducted with two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Data are presented as means and standard deviations. *p < 0.05, **p < 0.01; analyzed using a two-way analysis of variance followed by Tukey’s multiple comparison test.

Article Snippet: The STR/ort mice were administered tail-vein injections of 1 μg/mouse anti-EDIL3 IgG1 (MABS1976; MilliporeSigma), 2 μg/mouse recombinant EDIL3 protein (6046-ED; R&D Systems), 2 μg/mouse CD98 protein (50813-M07H; Sino Biological, China), 1 μg/mouse isotype-matched control IgG1 monoclonal antibody (mAb) (mabg1-ctrlm; InvivoGen, USA), or vehicle PBS solution.

Techniques: Control, Micro-CT, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) inhibition promotes pro-inflammatory cytokine production in STR/ort mice. a) The variable expressions of 48 cytokines among vehicle control, isotype IgG1, EDIL3 IgG1, recombinant EDIL3 protein, and CD98 protein groups were identified by comparing the expression pattern in the serum of STR/ort mice and those with vehicle control group by heat-map analysis. Arrows indicate representative anti-inflammatory cytokines (red) and pro-inflammatory cytokines (green). b) Quantitation of each cytokine demonstrated serum samples from different groups using Luminex xMAP technology. Six anti-inflammatory cytokines and 20 pro-inflammatory cytokines were identified with the most significant difference between the five groups. c) Notably, many pro-inflammatory cytokines in serum increase with ageing, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GRO-alpha. EDIL3 antibody significantly promoted the increase of these pro-inflammatory cytokines. Analyses were conducted with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for selected pairs of groups. Data are presented as means and standard errors. *p < 0.05, **p < 0.01, ***p < 0.001. Data presented in Figure 5c were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. IgG, immunoglobulin G.

Article Snippet: The STR/ort mice were administered tail-vein injections of 1 μg/mouse anti-EDIL3 IgG1 (MABS1976; MilliporeSigma), 2 μg/mouse recombinant EDIL3 protein (6046-ED; R&D Systems), 2 μg/mouse CD98 protein (50813-M07H; Sino Biological, China), 1 μg/mouse isotype-matched control IgG1 monoclonal antibody (mAb) (mabg1-ctrlm; InvivoGen, USA), or vehicle PBS solution.

Techniques: Inhibition, Control, Recombinant, Expressing, Quantitation Assay, Luminex, Comparison