Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Knockdown of MLKL alleviated IVDD in TSZ stimulation model of NPCs. (A) Western blot analysis showing MLKL knockdown efficiency using sh1-MLKL, sh2-MLKL, and sh3-MLKL in NPCs (n = 3 per group). (B, C) Representative fluorescence images and quantification of MLKL expression in Control and sh-MLKL groups (n = 3 per group; scale bar = 50 μm). (D) PCR analysis of ACAN and MMP3 expression in NPCs from Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group). (E, F) Western blot analysis of ACAN and MMP3 protein levels in Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group). (G, H) Representative fluorescence images and quantification of ACAN and MMP3 expression in Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group; scale bar = 50 μm).∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Knockdown, Western Blot, Fluorescence, Expressing, Control

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Conditional knockout of Mlkl inhibited the process of IVDD in mice. A) Schematic diagram of Mlkl CKO mouse generation. (B, C) Schematic illustrations of the LSI surgery model and the aging mouse model. (D, E) Representative IVD sections stained with H&E and SF from WT and Mlkl CKO groups after 8 weeks of LSI exposure (n = 5 per group; scale bars = 200 μm and 50 μm). (F, G) Representative IVD sections stained with H&E and SF from WT and Mlkl CKO groups in the aging model (n = 5 per group; scale bars = 200 μm and 50 μm). (H) Representative fluorescence images and quantification of Acan and Mmp3 expression in WT and Mlkl CKO groups following LSI (n = 5 per group; scale bars = 200 μm and 50 μm). (I) Representative fluorescence images and quantification of Acan and Mmp3 expression in WT and Mlkl CKO groups in the aging model (n = 5 per group; scale bars = 200 μm and 50 μm).

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Knock-Out, Staining, Fluorescence, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: USP39 alleviated IVDD by inhibiting ubiquitination and phosphorylation of MLKL. (A, B) Western blot analysis of USP39 knockdown efficiency (n = 3 per group). (C, D) Western blot analysis of USP39 overexpression efficiency (n = 3 per group). (E) Western blot analysis of MLKL ubiquitination and phosphorylation levels in Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (F) PCR analysis of ACAN and MMP3 expression in NPCs from Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (G, H) Western blot analysis of USP39, ACAN, and MMP3 expression in Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (I) Representative immunofluorescence staining and fluorescence intensities of ACAN and MMP3 in NPCs from Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group; scale bar = 50 μm). (J) Expression levels of ACAN and MMP3 in Control, TSZ, TSZ + sh-USP39, TSZ + sh-USP39+sh-MLKL, and TSZ + sh-USP39+NSA groups (n = 3 per group). (K) Representative immunofluorescence staining and fluorescence intensities of ACAN and MMP3 in NPCs from Control, TSZ, TSZ + sh-USP39, TSZ + sh-USP39+sh-MLKL, and TSZ + sh-USP39+NSA groups (n = 3 per group; scale bar = 50 μm). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Overexpression of Usp39 attenuated in vivo IVDD progression by suppressing MLKL phosphorylation. (A) Schematic illustration of AAV-Usp39 injection following LSI. (B) RT-qPCR analysis confirming significantly increased Usp39 mRNA levels in NP tissues following AAV-Usp39 injection. (C) Representative lumbar spine MRI of mice at 8 weeks in Sham (n = 5), LSI (n = 5), and LSI + AAV-Usp39 (n = 5) groups. (D, E) Representative H&E and SF staining of spinal tissues from Sham, LSI, and LSI + AAV-Usp39 groups (n = 5 per group; scale bars = 200 μm and 50 μm). (F–H) Representative immunofluorescence staining of Acan, Mmp3, and p-Mlkl in Sham, LSI, and LSI + AAV-Usp39 groups (n = 5 per group; scale bars = 200 μm and 50 μm).

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Over Expression, In Vivo, Phospho-proteomics, Injection, Quantitative RT-PCR, Staining, Immunofluorescence

Journal: Frontiers in Neuroanatomy

Article Title: Transmitter and ion channel profiles of saccadic omnipause neurons and cholinergic non-omnipause neurons in human nucleus raphe interpositus

doi: 10.3389/fnana.2025.1670220

Figure Lengend Snippet: Immunoperoxidase-based histochemical classification of omnipause neurons (OPN) and cholinergic non-OPNs in human nucleus raphe interpositus (RIP). (A) Combined immunoperoxidase labeling of perineuronal net (PN) marker aggrecan (ACAN, black) and non-phosphorylated neurofilament marker SMI32 (brown) reveals OPNs that are arranged as two columns around the midline in human RIP. The box indicates the area illustrated at higher magnification in B. (B) Close-up demonstrating OPNs co-immunolabeled with ACAN and SMI32 (red arrows) Note the rather dispersed arrangement of OPNs around the midline compared to OPNs in monkey (C) . Also note several neurons that are not ensheathed by PN marker ACAN but express SMI32-immunolabelling (green arrows). (C) In monkey RIP, combined peroxidase labeling of PN marker ACAN (black) and ChAT (brown) demonstrates the absence of such cholinergic neurons and tightly organized OPN columns around the midline. (D) Combined peroxidase labeling of perineuronal net (PN) marker aggrecan (ACAN, black) and non-phosphorylated neurofilament marker SMI32 (brown) and PN marker CSPG (black) choline acetyltransferase (ChAT, brown) reveal the cholinergic non-OPN population (green arrow) that are not ensheathed by PNs. Scale bar represents 1 mm A , 100 μm B,C and 50 μm in D .

Article Snippet: ACAN , Mouse/Monoclonal , Aggrecan , Acris Antibodies GmbH, 32052 Herford, GERMANY , AB_972582 , 1:75.

Techniques: Labeling, Marker, Immunolabeling

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-AGC1 (D5I6I) , Cell Signaling Technology , Cat#64169.

Techniques: Recombinant, In Vitro, In Vivo, Cloning, Plasmid Preparation, Software, CRISPR

Journal: Spine

Article Title: Pingyangmycin-induced in vivo lumbar disc degeneration model of rhesus monkeys.

doi: 10.1097/BRS.0000000000000736

Figure Lengend Snippet: Figure 6. Immunohistochemistry assay for MMP-3, type II collagen and aggrecan in the nucleus pulp- osus of the 3 groups ( × 400). A–C , The number of cells staining positive for MMP-3 in the nucleus pulposus had increased compared with the Sham and Vehicle control groups. D–I , the number of cells staining positive for type II collagen and aggrecan in the nucleus pulposus had decreased compared with the Sham and Vehicle control groups, respectively. MMP indicates matrix metalloprotease; PYM, Ping- yangmycin.

Article Snippet: SPINE140699_LR E202 23/01/15 9:19 AM Spine www.spinejournal.com E203 metalloprotease (MMP)-3 (1:100, both from Bioss Inc., Beijing, China) or aggrecan (1:100; Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China).

Techniques: Immunohistochemistry, Staining, Control

Journal: Spine

Article Title: Pingyangmycin-induced in vivo lumbar disc degeneration model of rhesus monkeys.

doi: 10.1097/BRS.0000000000000736

Figure Lengend Snippet: Figure 8. mRNA expressions of ( A ) aggrecan, ( B ) Col2 α 1, ( C ) Col1 α 1, ( D ) MMP-3, ( E ) ADAMTS-5, ( F ) vWF, ( G ) TNF- α , ( H ) IL-1 β , and ( I ) IL-6 at 15 months after surgery. * P < 0.05 vs. Sham group; † P < 0.05 vs. Vehicle control group. MMP indicates matrix metalloprotease; mRNA, mes- senger RNA; PYM, Pingyangmycin; IL-1 β , interleukin-1 β ; ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; vWF, von Willebrand factor; TNF- α , tumor necrosis factor α .

Article Snippet: SPINE140699_LR E202 23/01/15 9:19 AM Spine www.spinejournal.com E203 metalloprotease (MMP)-3 (1:100, both from Bioss Inc., Beijing, China) or aggrecan (1:100; Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China).

Techniques: Control