Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: The CD30 ligand isoforms have distinct C-terminal extracellular regions. ( A ) Protein alignment of the two isoforms using Clustal Omega. Red—shared intracellular domain; cyan—shared transmembrane domain; purple—shared extracellular domain; black—predicted alpha helix within shared extracellular domain; yellow—extracellular sequences unique to each isoform. Asterisks indicate matching amino acid identity, dots and double dots indicate similarity, and hyphen indicates absence of residues in Iso2. The N-terminus for both proteins is intracellular and identical through residue 136, which includes the transmembrane domain at residues 38–62. The residues following 136 are distinct extracellular C-termini. ( B ) The predicted structural homology of CD30L isoforms to known protein structures generated by the Phyre2 web portal. Iso1 ( left ) showed strong homology to human TNF-alpha with several antiparallel beta-sheets. However, the software did not predict the presence of strong secondary structures for Iso2 ( right ), instead matching homology to small portions of proteins with unstructured domains. The two isoforms did share a predicted alpha helix that was membrane proximal on the extracellular side, present on the left side of each image.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Residue, Generated, Software, Membrane

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Both CD30L isoforms were expressed at the RNA level in primary human cells. ( A ) PBMCs were isolated from healthy donors ( n = 6). Cells were either untreated (nt) or stimulated with DMSO, PMA/ionomycin (P/I), lipopolysaccharide (LPS) isolated from E . coli 0111:B4 (LPS), anti-CD3/CD28 beads (CD3/28), or immune complex (IC) for 6 or 24 h. The graphs show actin-normalized expression of both isoforms at both timepoints for all stimulation conditions. Single asterisks indicate p ≤ 0.05 and double asterisks indicate p ≤ 0.01 between the conditions indicated. Error bars represent standard deviation between donors. ( B ) PBMCs were isolated from healthy donors ( n = 3, half of the 6 total donors). Cells were either untreated (nt) or stimulated with PMA/ionomycin (P/I) or immune complex (IC) for 24 h. After incubation, cells were subject to flow cytometry for B cell marker CD19, monocyte marker CD14, and T cell marker CD3, as well as CD30 ligand Iso1. PBMC cell types were present at expected ratios. Error bars are standard deviation between donors.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Isolation, Expressing, Standard Deviation, Incubation, Flow Cytometry, Marker

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 has a significantly impaired ability to bind CD30 compared to Iso1, but both are present in an exogenous CD30 protein complex. ( A ) HEK cells were transfected with vector (vec) control or FLAG-tagged Iso1 (1FL) or Iso2 (2FL). Lysates and supernatants were subject to a plate-based binding assay with CD30 coated on the plates and anti-FLAG antibody was used for detection. ( B ) Untagged Iso1 (1NT) was transfected into HEK cells with or without FLAG-tagged Iso2 (2FL), as well as 2FL alone. Cell lysates were subject to a plate-based binding assay. An anti-Iso1-specific CD30L antibody was used for capture and an anti-FLAG antibody for detection. ( C ) HEK cells were transfected with increasing amounts of 1NT plasmid, with either vector (vec, undetectable) control or 2FL at a constant amount and lysates were subject to a plate-based binding assay with recombinant CD30 coated on the plates. Anti-FLAG antibody was used for the detection of 2FL. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso1 induces cytokine production in co-culture with CD30+ cells while Iso2 has no effect. Increasing quantities of HEK cells were transfected with either vector (vec) or CD30L isoforms with (FL) or without (NT) FLAG tag. After transfection, K299 cells were co-cultured with HEK cells for 24 h. Supernatants were harvested and subject to cytokine analysis by MSD assay. Error bars for all are from standard deviations between technical replicates.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, FLAG-tag, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 partially blocks Iso1-mediated stimulation of K299 cells. ( A ) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. ( B , C ) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity ( B ) and IL-6 production ( C ) were measured. ( D ) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. ( E ) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay, Expressing, Flow Cytometry, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. ( A ) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. ( B ) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. ( C , D ) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction ( C ), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads ( D ). Both were subject to SDS-PAGE and Western blot. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Recombinant, Incubation, SDS Page, Western Blot

Journal: Molecular Cancer

Article Title: Targeting CD30L in B-cell non-Hodgkin lymphoma: novel peptide conjugates and their therapeutic potential

doi: 10.1186/s12943-025-02393-9

Figure Lengend Snippet: TG-1 suppresses CD30-CD30L-mediated B-NHL proliferation by targeting the non-canonical NF-κB pathway. Relative cell viability of Jeko-1 ( A ) and Raji ( B ) cells was assessed following incubation with 5 and 10 µg/ml of CD30, 10 µg/ml of CD30 pretreated with 25 µM TG-1, or 10 µg/ml of CD30 pretreated with 1 µg/ml CD30L antibodies for 4–6 h at 37 °C, as determined by MTT assays. ( C ) Relative cell viability of Granta-519 cells was evaluated after exposure to 10 µg/ml of CD30 that had been pretreated with the CD30L plasmid (1 µg), as well as with CD30 alone, using MTT assays. The expression levels of NFκB-2, IKKα, phosphorylated IKKα, and RELB in Jeko-1 ( D ) and Raji ( E ) cells were evaluated using Western blot analysis after treatment with 10 µg/ml CD30, preceded by pretreatment with either 25 µM TG-1 or1 µg/ml CD30L antibodies. ( F , G ) The protein expression levels of NFκB-2, IKKα, phosphorylated IKKα, and RELB in D and E were quantified using ImageJ. GAPDH served as an internal control. ( H-O ) Immunofluorescence was used to measure the expression levels of NFκB-2 and RELB in Jeko-1 ( H and I ) and Raji ( L and M ) cells following treatment with 10 µg/ml CD30, with a preceding pretreatment of either 25 µM TG-1 or1 µg/ml CD30L antibodies. Scale bar = 20 μm. ( J , K , N , and O ) The normalized fluorescence intensity for H , I , L , and M , respectively. ( P-S ) The relative cell viability of Jeko-1 ( P ), Raji ( Q ), Su-4 ( R ), and Granta-519 ( S ) cells was evaluated after exposure to combinations of 10 µg/ml CD30 and 200 nM IKK16, as determined by MTT assays. Data shown in this figure are the mean values (± SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (# P ≥ 0 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Treatments included 25 μM TG-1 or Random peptides, recombinant human CD30 protein (HY-P70023, MedChemExpress, NJ, USA) at varying concentrations, and IKK16 (0-500 nM; HY-13687, ChemExpress, China), either alone or in combination, for 24 h at 37 °C.

Techniques: Incubation, Plasmid Preparation, Expressing, Western Blot, Control, Immunofluorescence, Fluorescence

Journal: Molecular Cancer

Article Title: Targeting CD30L in B-cell non-Hodgkin lymphoma: novel peptide conjugates and their therapeutic potential

doi: 10.1186/s12943-025-02393-9

Figure Lengend Snippet: Schematic illustration of the interaction between TG-1-DKK, F-TG-1-AuNPs, and F-TG-1-AuNPs + DOX with CD30L-positive B-NHL cells. Our in vitro experiments have shown that CD30 interacts with CD30L on B-NHL cells, resulting in the promotion of B cell proliferation and the suppression of cell death. Nanoparticles functionalized with the TG-1 peptide selectively bind to CD30L. This binding disrupts the CD30-CD30L interaction, inhibiting CD30L-mediated reverse signaling and leading to a decrease in proliferation. Furthermore, the conjugation of TG-1 with the anti-cancer peptide DKK enhances cell lysis by targeting CD30L. In vivo investigations have demonstrated that FITC-labeled TG-1 can identify and inhibit tumor cell proliferation through the bloodstream. Additionally, F-TG-1-AuNPs + DOX effectively inhibit tumor growth in vivo

Article Snippet: Treatments included 25 μM TG-1 or Random peptides, recombinant human CD30 protein (HY-P70023, MedChemExpress, NJ, USA) at varying concentrations, and IKK16 (0-500 nM; HY-13687, ChemExpress, China), either alone or in combination, for 24 h at 37 °C.

Techniques: In Vitro, Binding Assay, Conjugation Assay, Lysis, In Vivo, Labeling