Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

Techniques: Expressing, Clinical Proteomics, Western Blot, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Ubiquitin Proteomics, Mutagenesis, Control

Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: Impact of SDE2 knockdown on cellular metabolism and autophagy in MM cells. (A) Fluorescence imaging of CFDA/DAPI-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (B) Fluorescence imaging of acridine orange-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (C) MitoTracker staining to evaluate mitochondrial activity in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (D) Transmission electron microscopy (TEM) images showing autophagosomes (indicated by red arrows) and autolysosomes (indicated by yellow arrows) in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (E) Western blot analysis of p62 and ATG5 expression levels in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (F) mRFP-GFP-LC3 dual fluorescence labeling of OPM-2 and KMS-11 cells with or without SDE2 knockdown, visualizing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). The bar graphs quantify the total number of puncta and the relative proportion of autophagosomes to autolysosomes. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

Techniques: Knockdown, Fluorescence, Imaging, Staining, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Labeling

Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: SDE2 promotes MM cell proliferation via its UBL domain–dependent inhibition of ferroptosis and autophagy. (A) Colony formation assays were performed in H929 cells transfected with empty vector, SDE2, or SDE2Δ1. Representative images (left) and quantification of colony numbers (right) are shown. (B) Intracellular glutathione (GSH), ferrous iron (Fe 2+ ), and malondialdehyde (MDA) levels were measured using corresponding colorimetric kits. (C) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in the indicated groups; GAPDH was used as a loading control. Densitometric quantification is shown on the right. (D) Lipid peroxidation was assessed by BODIPY 581/591 C11 staining in H929 cells expressing vector, SDE2, or SDE2Δ1. Representative fluorescence images of oxidized (green) and non-oxidized (red) signals are shown. (E) Quantification of oxidized BODIPY fluorescence intensity from (D). (F) Quantification of MitoTracker Red fluorescence intensity in the indicated groups. (G) Mitochondrial morphology was visualized by MitoTracker Red staining in H929 cells. (H) Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent-tagged reporter system. Representative images show red (mRFP), green (GFP), and merged puncta. (I) Quantification of LC3 puncta number and percentage of mRFP + /GFP − autolysosomes per cell from (H). (J) Western blot analysis of autophagy-related proteins p62, ATG5, and ATG7 in the indicated groups. GAPDH served as a loading control. Densitometric quantification is shown below. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

Techniques: Inhibition, Transfection, Plasmid Preparation, Western Blot, Control, Staining, Expressing, Fluorescence

Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: Modulation of autophagy and ferroptosis in MM cells through SDE2 manipulation and pharmacological interventions. KMS-11 cells treated with SDE2 knockdown and/or the autophagy inhibitor chloroquine (CQ): (A) Western blot analysis of autophagy-related proteins LC3 and p62 in KMS-11 cells. Densitometric quantification is shown on the right. (B) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells under the same treatments as in (A), with corresponding quantification on the right. (C) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. H929 cells treated with SDE2 overexpression and/or the ferroptosis inducer Erastin: (D) Western blot analysis of autophagy-related proteins LC3 and p62 in H929 cells. Densitometric quantification is shown on the right. (E) Western blot analysis and quantification of ATG5 and ATG7 in H929 cells under the same treatments as in (D). (F) mRFP-GFP-LC3 dual fluorescence labeling visualizing autophagic flux in H929 cells, showing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). KMS-11 cells treated with SDE2 knockdown and/or ATG5 knockdown: (G) Western blot analysis and quantification of Beclin1 and p62 in KMS-11 cells expressing shCtrl or shSDE2, with or without ATG5 knockdown. (H) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells with single or combined knockdown of SDE2 and ATG5. Quantification is shown on the right. (I) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

Techniques: Knockdown, Western Blot, Over Expression, Fluorescence, Labeling, Expressing

Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: Functional interplay between ATG5 and SDE2 in MM progression and ferroptosis regulation. (A) Western blot analysis of ATG5 expression in malignant cells (T) and non-malignant bone marrow cells (N) isolated from the peripheral blood of MM patients. (B) Western blot analysis confirming the efficiency of ATG5 overexpression in OPM-2 and H929 cells transfected with a plasmid encoding the full-length ATG5 sequence. (C) Crystal violet staining of Transwell migration assays comparing the migratory ability of OPM-2 and H929 cells with or without ATG5 overexpression. (D) Experimental setup for H929 cells treated with SDE2 overexpression, ATG5 overexpression, or a combination of both. (E) Western blot analysis of SDE2 and ATG5 protein levels in H929 cells from the four groups defined in (D). GAPDH was used as a loading control. (F) (Top) Colony formation assay evaluating the proliferative capacity of H929 cells under different treatments. (Bottom) Transwell assay assessing the migratory ability of H929 cells under different treatments. (G) Biochemical analysis of ferroptosis-related markers (MDA, GSH, and Fe 2+ ) in H929 cells subjected to different treatments. (H) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in H929 cells under different treatments. (I) Western blot analysis showing the effects of different treatments on LC3 expression and p62 degradation in H929 cells. (J) Representative images of tumors formed 35 days after subcutaneous inoculation of differently treated H929 stable cell lines into nude mice. (K) Tumor growth curves showing volume changes of tumors derived from differently treated H929 cell lines over time. (L) Average tumor weights of tumors derived from differently treated H929 cell lines. (M) Western blot analysis of NCOA4, ACSL4, FTH1, and GPX4 expression levels in tumors derived from differently treated H929 cell lines. (N) Western blot analysis showing the activation levels of the ATG5/ATG7 pathway and p62 degradation in tumors derived from differently treated H929 cell lines. (O) Representative images of H&E staining and Ki67 immunohistochemical staining of tumor sections derived from differently treated H929 cell lines. The bar graph quantifies the percentage of proliferating cells (Ki67-positive). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

Techniques: Functional Assay, Western Blot, Expressing, Isolation, Over Expression, Transfection, Plasmid Preparation, Sequencing, Staining, Migration, Control, Colony Assay, Transwell Assay, Stable Transfection, Derivative Assay, Activation Assay, Immunohistochemical staining