Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

doi: 10.1074/jbc.m109.013185

Figure Lengend Snippet: FIGURE 2. Expression of the HAPLNs is highly reduced in malignant gliomas. A, comparison of mRNA expression levels for the members of the link protein (LP) and lectican families in different types of high grade gliomas using microarray data from the NCI Repository for Molecular Brain Neoplasia Data. Each column rep- resents the level of expression (-fold-over-control, mean S.E.) for each tumor type compared with normal brain tissue. Results for each gene were analyzed by one-way ANOVA (* indicates significant differences versus control, p 0.01 or lower). The results show strong reduction of the expression of HAPLN2 and HAPLN4 that contrasts with the up-regulation of brevican and versican in gliomas. BCAN, brevican; ACAN, aggrecan; NCAN, neurocan; VCAN, versican. Data were renormalized using neuronal markers to test the correlation of HAPLN2 and HAPLN4 to the abundance of neurons in the tumor parenchyma (see supplemental Fig. 1). B, total homo- genates from high grade gliomas (two grade III astrocytomas and seven grade IV glioblastomas, of which five are shown) and age-matched controls were probed for HAPLN1, HAPLN2, HAPLN4, and actin as loading con- trol. C, quantification of normalized results from B; HAPLN2 and HAPLN4 are expressed at very low levels in the tumor parenchyma (white bars) compared with normal brain (black bars). IOD, integrated optical density. D, detection of HAPLN2 and HAPLN4 by RT-PCR using total RNA from low passage cultures of human fetal astrocytes. The same procedure yielded negative results with five different human glioma cell lines tested (U87MG, U118MG, U251MG, U373MG, and A172, not shown). RT, presence () or absence () of reverse transcriptase in the mixture. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. E, detection of HAPLN4 by Western blot in the conditioned medium from primary glioma-derived neurospheres (identified as G2 to G12). Cultures with detectable expression of HAPLN4 also expressed high levels of brevican (not shown). BSA, bovine serum albumin.

Article Snippet: Human HAPLNs were detected with mouse monoclonal antibodies against HAPLN1 (clone 8A4, NICHDDevelopmental StudiesHybridomaBank, IowaCity, IA), HAPLN2 (Abnova, Taiwan) and HAPLN4 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Comparison, Microarray, Control, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Western Blot, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

doi: 10.1074/jbc.m109.013185

Figure Lengend Snippet: FIGURE 5. HAPLN4 does not associate to the surface of cultured glioma cells. A, subcellular fractionation of human adult brain cortex followed by probing of the nuclear-enriched (n), membrane-enriched (m), and soluble (s) fractions with antibodies against versican (VCAN), brevican (BCAN), HAPLN1, HAPLN2, and HAPLN4. Only HAPLN4 appeared tightly associated to mem- brane-containing fractions, whereas all the other ECM proteins distributed in the soluble and particulate fractions. B, transient transfection of HPLN4 in cul- turedhuman(U87MGandU251MG)andrat(CNS-1)gliomacelllinesfollowedby preparation of conditioned medium (CM) and total cell lysates (Ly) for Western blotting; equal amounts of total protein (10 g) were loaded for all samples. HAPLN4appearedlargelysecretedtothemedium,andlittleproteinwasfoundin thecelllysates.C,U251MGcells,transientlytransfectedwithbrevicanorHAPLN4, wereculturedonglasscoverslipsandprocessedforimmunofluorescenceaspre- viously described (22). Primary antibodies were added to unfixed, unpermeabi- lizedcells(Livestaining)ortopost-fixedandpermeabilizedcells(Post-fixed).Both proteins were detected in the cells after fixation, but only brevican was detected associated to the cell surface of non-permeabilized cells. D, U251MG cells expressing HAPLN4 alone (none) or co-expressed with brevican (BCAN), HAS2, or brevicanHAS2 (BCANHAS2), were collected and processed for Western blot- ting as in B. None of the co-expressed proteins significantly affected the distribu- tion of secreted HAPLN4. Brevican accumulated in the cell lysates in presence of HA synthesized by HAS2; formation of HA pericellular coats after HAS2 transfec- tion was verified by particle-exclusion assay (seesupplemental Fig. 2). Equal pro- tein loading in culture media and lysates was controlled by detection of total albumin in the medium and actin in the cell lysates. Western blots show more intense bands than in B because they were developed by quantitative chemilu- minescence and exposed for maximum detection of each protein. BSA, bovine serum albumin.

Article Snippet: Human HAPLNs were detected with mouse monoclonal antibodies against HAPLN1 (clone 8A4, NICHDDevelopmental StudiesHybridomaBank, IowaCity, IA), HAPLN2 (Abnova, Taiwan) and HAPLN4 (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Fractionation, Membrane, Transfection, Western Blot, Expressing, Synthesized, Exclusion Assay

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

doi: 10.1074/jbc.m109.013185

Figure Lengend Snippet: FIGURE 6. HAPLN4 is strongly associated to cell membranes of neural tissue. A, total membranes (M) obtained from subcellular fractionation of adult human brain cortex were resuspended in 25 mM Tris-HCl buffer, pH 7.4 (Tris), Tris buffer containing 10 mM EDTA (EDTA), Tris buffer containing 0.5% w/v Triton X-100 (Tx100), or 100 mM sodium carbonate buffer, pH 11.0 (CO3). After incubation, membranes were centrifuged, and the resulting superna- tant (s) and pellet (p) were processed for Western blotting. To verify whether released HAPLN4 was effectively water-soluble, membranes were extracted with 2% Triton X-114 as indicated under “Experimental Procedures” and sub- sequentlyseparatedbytemperature-dependentpartitioninaninsolublepel- let (Ins), aqueous phase (Aq), and Triton-containing phase (Tx). All the results suggested that HAPLN4 was strongly, but peripherally, associated to cell membranes. B, brain cortex membranes resuspended in Tris-HCl buffer con- taining 10 mM sodium acetate were treated with chondroitinase ABC (CHase) or chondroitinase plus phosphatidylinositol-specific phospholipase C (PLC). Afterincubation,membraneswereprocessedasindicatedinA.C,braincortex membranes resuspended in Tris-HCl buffer were treated with hyaluronidase (HAse) for 4 h, separated into supernatant (s) and pellet (p) fractions as indi- cated, and processed for Western blotting for versican (VCAN), brevican (BCAN), HAPLN4, and HAPLN1. Only HAPLN4 was insensitive to hyaluronidase treatment.

Article Snippet: Human HAPLNs were detected with mouse monoclonal antibodies against HAPLN1 (clone 8A4, NICHDDevelopmental StudiesHybridomaBank, IowaCity, IA), HAPLN2 (Abnova, Taiwan) and HAPLN4 (R&D Systems, Minneapolis, MN).

Techniques: Fractionation, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Identification of HAPLN1 as a factor capable of inducing NF-κB activity in myeloma cells. A, SDS-PAGE and Coomassie Blue staining of the enriched fractions with NF-κB–inducing activity previously identified by Markovina et al. (35). Three bands (*) from fraction 3 (F3) that contained the highest NF-κB–inducing activity were excised and analyzed by nano-LC-MS/MS. Shown are identified factors with the number of unique peptides identified in parentheses. B, EMSA analysis of RPMI8226 cells incubated with 10 ng/ml TNFα for 15 min or CM collected from HEK293 transiently transfected with vector containing C-terminally MYC-tagged proteins: calumenin, osteonectin, or galectin-3–binding protein (Gal-3BP) for 2 h. Results are representative of at least three independent experiments. C, representative immunoblot analysis (IB), using anti-MYC antibody, of CM from HEK293 cells transiently transfected with calumenin, osteonectin, or galectin-3–binding protein expression vector showing the presence of corresponding secreted factors. D, immunoblot analysis, using anti-MYC antibody, of HAPLN1 expression in CM collected from transiently transfected HEK293 cells with the HAPLN1 expression vector positive for induction of NF-κB activity. Smaller HAPLN1 (H1) fragments are indicated *, larger immunoreactive bands. E, EMSA analysis of RPMI8226 cells treated with CM from transiently transfected HEK293 cells containing HAPLN1 for 2 h in the absence or presence of 100 nm bortezomib (Bort). Induced NF-κB activity is highly variable in more than five independent experiments, possibly dependent on the variability of smaller H1 species seen in D.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Activity Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Incubation, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Generation and characterization of recombinant HAPLN1 proteins. A, diagram of HAPLN1 domains. The numbers indicate the amino acid positions. SP, signal peptide; IG, immunoglobulin-like domain; PTR1, proteoglycan tandem repeat 1 domain; PTR2, proteoglycan tandem repeat 2 domain. B, SDS-PAGE and GelCodeTM staining of GST-fused HAPLN1 domain proteins. Full-length (FL) HAPLN1 (not GST-tagged) is from R&D. *, band of interest. C (top), representative EMSA analysis of NF-κB and Oct-1 activities in RPMI8226 cells following incubation with FL-H1, GST-fused H1-IG, H1-P1, and H1-P2 at 100 nm for 4 h or TNFα (10 ng/ml) for 15 min. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. (error bars) of three independent EMSA experiments shown above. *, p < 0.05; **, p < 0.01 when compared with appropriate control (untreated or GST only). D, representative EMSA analysis of three independent experiments where RPMI8226 cells were stimulated with increasing doses of lipopolysaccharide (LPS) for 1 h or 10 ng/ml TNFα for 15 min. E, representative graph of three independent endotoxin quantifications using the Pierce LAL chromogenic endotoxin quantification kit (Thermo Fisher Scientific). Depicted is the span of the average amount of endotoxin found in three independent H1-P1 purifications. The eluted fractions were further diluted 50–100-fold when added to culture media for NF-κB activation analyses, making the levels of contaminating lipopolysaccharide at least 3 orders of magnitude below what is detectable for NF-κB activation by EMSA.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Recombinant, SDS Page, Staining, Incubation, Activation Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 domain activates NF-κB in myeloma cells. A (top), representative EMSA analysis of RPMI8226 cells incubated with increasing concentrations of H1-P1 for 2 h. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. (error bars) of three independent EMSA experiments (above). *, p < 0.05; **, p < 0.01 when compared with untreated (0 nm). B (top), representative EMSA analysis of RPMI8226 cells incubated with 100 nm H1-P1 for the times indicated. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. of three independent EMSA experiments (above). *, p < 0.05; **, p < 0.01 when compared with unstimulated 1-h lane. C, supershift analysis of RPMI8226 cells treated with 100 nm H1-P1 for 2 h using antibodies against the five NF-κB family members. Results are representative of three independent experiments. D, SDS-PAGE and GelCodeTM staining of GST-fused H1-P1 following incubation with GST-fused PreScission protease. H1-P1c, cleaved H1-P1 fragment. E, EMSA analysis of RPMI8226 cells pretreated for 30 min with 100 nm bortezomib as indicated and stimulated with 100 nm H1-P1c or 10 ng/ml TNFα for the times indicated, representative of two independent experiments. F, RPMI8226 cells treated with the indicated concentrations of H1(32–354) for 4 h and analyzed by EMSA, representative of two independent experiments. G, RPMI8226, MM.1S, and H929 cell lines were incubated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 4 h and assayed for induction of NF-κB activity by EMSA, representative of three independent experiments. Where indicated, EMSA was done in the presence of a RelB antibody (RelB Aby) to shift RelB complexes to enable quantification of canonical NF-κB activation. -Fold change of NF-κB DNA binding as measured by phosphor image quantification, corrected for Oct-1 DNA binding control, and normalized to unstimulated but RelB antibody–treated, is indicated below the gel.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Incubation, Activation Assay, SDS Page, Staining, Activity Assay, Binding Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Hyaluronic acid binding is unnecessary for HAPLN1-PTR1–induced NF-κB activation. A, EMSA analysis of NF-κB activity in RPMI8226 cells following incubation with low-molecular weight HA (LMW-HA; 29 kDa) or high molecular weight HA (HMW-HA; 289 kDa) at the microgram amounts indicated for 2 h. Results are representative of two independent experiments. B, EMSA analysis of RPMI8226 cells treated with 100 nm H1-P1, low-molecular weight HA, high-molecular weight HA, or hyaluronidase (HYAL) in the amounts indicated for 4 h. C, H1-P1 HA-binding mutant (H1-P1 HABD mt) was made by mutating the critical residues necessary for HA binding within H1-P1 to alanine where illustrated. RPMI8226 cells were incubated with the indicated concentrations (nm) of recombinant GST-fused H1-P1 HABD mt for 4 h and assayed by EMSA. Results are representative of three independent experiments. -Fold change of NF-κB activity was determined as described previously.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Binding Assay, Activation Assay, Activity Assay, Incubation, Molecular Weight, High Molecular Weight, Mutagenesis, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 causes bortezomib-resistant NF-κB activation. A, representative EMSA analysis of RPMI8226 cells incubated with 10 ng/ml TNFα or 100 nm H1-P1 in the absence or presence of increasing concentrations (nm) of bortezomib (Bort). B, graph depicts the mean ± S.D. (error bars) of the quantification of three independent replicates of EMSA analysis as in A. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, the proteasome activity in RPMI8226 cells was analyzed following treatment with increasing doses of bortezomib (nm) in the presence and absence of 100 nm H1-P1 using a Proteasome-Glo cell-based assay (Promega) and plotted using the basal proteasome activity in 0 nm bortezomib set as 1. Results represent mean ± S.D. of three independent experiments. D, EMSA analysis of RPMI8226 cells incubated with increasing concentrations of lactacystin (μm) and stimulated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 4 h. Results are representative of two independent experiments. E, EMSA analysis of RPMI8226 cells pretreated with 100 nm bortezomib for 30 min and stimulated with increasing doses of TNFα (ng/ml) for 15 min. -Fold change of NF-κB activity was determined as described previously.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Activation Assay, Incubation, Activity Assay, Cell Based Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 causes bortezomib-resistant IκBα degradation. A, representative immunoblot analysis of IκBα, IκBβ, and tubulin in RPMI8226 cells pretreated for 30 min with cycloheximide (Cx; 20 μg/ml) or bortezomib (Bort) (100 nm) and stimulated with H1-P1 (100 nm) where indicated. B, three biological replicates of degradation of IκBα and IκBβ as in A were quantified and plotted with mean ± S.D. (error bars). *, p < 0.05. C, immunoblot analysis of RPMI8226 cells pretreated for 30 min with 100 nm bortezomib where indicated and stimulated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 2 h. The top gel image is a longer exposure of the IκBα blot. The positions of IκBα, phospho-IκBα, and IκBα ubiquitin ladders are indicated. Results are representative of at least three independent experiments. D, RPMI8226 cells pretreated for 30 min with 10 μm IKK16 and 20 μg/ml cycloheximide were stimulated with H1-P1 for the indicated times and analyzed by EMSA and immunoblotted (IB) for indicated proteins. Results are representative of three independent experiments. E, EMSA and immunoblot analysis for the indicated proteins of RPMI8226 cells pretreated for 30 min with increasing concentrations MLN4924 (μm) and stimulated with 100 nm H1-P1 for 4 h or 10 ng/ml TNFα for 15 min and analyzed by EMSA and immunoblotting for the indicated proteins. F, results represent mean ± S.D. (*, p < 0.05; **, p < 0.01) of quantification of three independent EMSA and phospho-IκBα immunoblot analyses as in E at 100 μm MLN29424.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Western Blot, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1 is secreted by BMSCs, and HAPLN1-PTR1 causes bortezomib resistance in myeloma cells. A, mRNA expression of HAPLN1 in myeloma BMSCs analyzed by qRT-PCR in triplicate, normalized to GAPDH mRNA expression; the mean for each sample is presented as a box-and-whisker plot. n = 18 myeloma patient BMSCs. B, schematic illustrating the locations of epitopes for different HAPLN1 antibodies utilized. Dashed lines indicate the uncertainty of the specific epitope regions. C, immunoblot analysis of GST-fused HAPLN1 domains showing the epitope specificity of HAPLN1 antibodies utilized. Four parallel SDS-PAGE analyses and immunoblots were performed to independently determine the reactivity of each antibody. *, species of interest. D, immunoblot analysis of HAPLN1 expression from conditioned media collected from myeloma BMSCs for 48 h. *, HAPLN1 immunoreactivities detected by two independent anti-HAPLN1 antibodies, H-93 and HPA019482 (HPA). Results are representative of three biological replicates. Pt, patient. E, immunoblot analysis of HAPLN1 expression in myeloma patient BM plasma fractions with four different HAPLN1 antibodies detecting PTR1 and PTR2 (K-14 and C-14) and IG (H-93 and HPA019482) domains. One membrane was probed with K-14 and H-93, and a second membrane was probed with C-14 and HPA019482. *, HAPLN1 immunoreactivities detected. NS, non-specific. Results are representative of three independent replicates. F, the indicated myeloma cell lines were cultured at 104 cells (°, except for MM.1S at 5 × 104 cells and 3 nm bortezomib (Bort)) with 100 nm GST or 100 nm H1-P1 alone or in combination with 10 nm bortezomib. After 3 days, cell viability was measured by an MTT assay. Each dot represents the mean of biological replicates performed in triplicate. Bar graphs represent the mean of three biological replicates ± S.D. (error bars). *, p < 0.05; **, p < 0.01. G, CD138+ cells from patients were cultured at 1–1.5 × 104 cells with 100 nm GST or 100 nm H1-P1 alone or in combination with 10 nm bortezomib, as indicated. Following 24 h, cell viability was measured by an MTT assay. Each square represents a technical replicate, and the bar graph represents the mean ± S.D. of triplicates. **, p < 0.01.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Whisker Assay, Western Blot, SDS Page, Clinical Proteomics, Membrane, Cell Culture, MTT Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Schematic of proposed role of HAPLN1 in myeloma. MM-BMSCs secrete HAPLN1. Secreted proteases produced by MM-BMSCs, MM cells, or possibly other cell types in the tumor microenvironment process HAPLN1 into PTR1/PTR2-containing fragments to induce an atypical NF-κB signaling pathway in myeloma plasma B-cells leading to bortezomib-resistant survival of MM cells. This pathway involves IKK activation and IκBα phosphorylation, but polyubiquitination of IκBα is hardly detected (denoted by a lighter and shorter ubiquitin chain). In contrast, TNFα activates the canonical NF-κB pathway that is sensitive to bortezomib inhibition, and both IκBα phosphorylation and polyubiquitination are readily detectable.

Article Snippet: Reagents purchased include recombinant human TNFα (654205, EMD Millipore), lipopolysaccharide (L2880, Sigma-Aldrich), clasto -lactacystin β-lactone (154226-60-5, Sigma-Aldrich), bortezomib (S1013, Selleckchem), IKK16 (S2882, Selleckchem), MLN4924 (505477, Calbiochem), cycloheximide (C7698, Sigma-Aldrich), recombinant HAPLN1 (2608-HP, R&D Systems), low/high molecular weight hyaluronan (GLR001 and GLR004, R&D Systems), hyaluronidase (75790-208, ProSci), and GelCode TM Blue Stain (Thermo Fisher Scientific).

Techniques: Produced, Clinical Proteomics, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics, Inhibition

Journal: Biomolecules & therapeutics

Article Title: Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

doi: 10.4062/biomolther.2023.080

Figure Lengend Snippet: Fig. 1. Expression of HAPLN1 indifferent stages of hair growth cycle and HAPLN1 enhanced the proliferation of HHGMCs. (A) Immunohis- tochemical staining of frozen sections of skin tissue. HAPLN1 (yellow arrows) staining of biopsies taken from 7-week-old mice and 20-month-old mice kept in conventional conditions (original magnifications: 100× and 200×; n=6). (B) Localization and characterization of HAPLN1 in the hair follicles of mice during each phase of the hair cycle. Localization of HAPLN1 in the hair follicles during anagen, catagen and telogen phases (original magnification, 400×; n=6). (C) In situ hybridization detection of HAPLN1 mRNA in each stage of hair growth cycle. The tissue sections were stained with HAPLN1 RNA probe according to the standard procedures (original magnification, 400×; n=6). (D) HHGMCs were pretreated with rhHAPLN (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. The cell proliferation was analyzed with the CCK-8 assay (n=4). *p<0.05 versus corresponding TGF-β2 group. (E) Immunofluorescence staining of frozen sections of skin tissue. Localization of HAPLN1 in the hair growth cycle during anagen, catagen and telogen phases (original magnification, 400×; n=6).

Article Snippet: Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Techniques: Expressing, Staining, In Situ Hybridization, CCK-8 Assay, Immunofluorescence

Journal: Biomolecules & therapeutics

Article Title: Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

doi: 10.4062/biomolther.2023.080

Figure Lengend Snippet: Fig. 2. rhHAPLN1 and/or HA increased TβRII levels expression in HHGMCs. (A) The plot and relative quantification of the expression of TβRII in HHGMCs treated with various doses (0 ng/mL; 5 ng/mL; 10 ng/mL; 20 ng/mL) rhHAPLN1 for 24 h. (B) The plot and the relative quantification of the expression of TβRII in HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. (C) The plot and the relative quantification of the expression of TβRII in HHGMCs following HAPLN1 knock- down. (D) The plot and relative quantification of the expression of TβRII in HAPLN1 knockdown HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. (E) The plot and the relative quantification of the expression of TβRII and HAS2 in HHGMCs pretreated with 4-MU (0.5 mM) and/or rhHAPLN1 (25 ng/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h (n=3). *p<0.05, **p<0.01 versus corresponding control group (HA/HAPLN1 0 ng/mL) or siCTL group.

Article Snippet: Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Techniques: Expressing, Quantitative Proteomics, Knockdown, Control

Journal: Biomolecules & therapeutics

Article Title: Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

doi: 10.4062/biomolther.2023.080

Figure Lengend Snippet: Fig. 3. Lack of endogenous CD44 decreased TβRII levels expression in HHGMCs. (A) The plot and the relative quantification of the ex- pression of TβRII in CD44 following HAPLN1 knockdown. (B) The plot and the relative quantification of the expression of TβRII in CD44 knockdown HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h (n=3). *p<0.05, **p<0.01 versus corresponding siCTL group.

Article Snippet: Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Techniques: Expressing, Quantitative Proteomics, Knockdown

Journal: Biomolecules & therapeutics

Article Title: Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

doi: 10.4062/biomolther.2023.080

Figure Lengend Snippet: Fig. 4. rhHAPLN1 and/or HA increased TβRII levels expression in HHGMCs surface membrane. The plot and the relative quantifica- tion of the expression of TβRII in HHGMCs pretreated with rhHAP- LN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. Cell surface levels of TβRII were defined by labeling cells with sulpho-NHS-LC-biotin. Immunopre- cipitation study was performed using an antibody against biotin (n=3). **p<0.01 versus corresponding control group (HA/HAPLN1 0 ng/mL).

Article Snippet: Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Techniques: Expressing, Membrane, Labeling, Control

Journal: Biomolecules & therapeutics

Article Title: Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

doi: 10.4062/biomolther.2023.080

Figure Lengend Snippet: Fig. 5. rhHAPLN1 and/or HA activate non-canonical pathway of TGF-β2-ERK1/2 signaling. The plot and the relative quantification of the expression of phosphorylation of (A) Smad2, (B) c-Raf, MEK1/2 and ERK1/2 in HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 23 h prior to stimulation with TGF-β2 (2 ng/mL) for 1 h (n=3). **p<0.01, ***p<0.001 and ****p<0.0001 versus corresponding control group (HA/HAPLN1 0 ng/mL).

Article Snippet: Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Techniques: Quantitative Proteomics, Expressing, Phospho-proteomics, Control