Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a Quantitative real-time PCR (qPCR) analysis of Sema3b mRNA expression in sciatic nerves across postnatal development (P0, P7, P14, P28, and P60). n = 3 sciatic nerves per time point. b Transversal section of the P14 sciatic nerve showing that SEMA3B + SOX10 + MBP - SCs ensheath unmyelinated TUJ1 + axons. The arrowhead and arrow indicate SEMA3B + SOX10 + MBP - nmSCs and SEMA3B - SOX10 + MBP + mSCs, respectively. The experiment was repeated 3 times on 3 mice. c , d Representative TEM images of the sciatic nerve from P60 control and Sema3b ∆SC mice. nmSC lamellipodia are pseudo-colored to highlight their locations. Asterisks denote adjacent axons and circles denote partially wrapped axons. Arrowheads indicate lamellipodia protruding into the endoneurial space. The experiment was repeated 3 times on 3 mice. e , f Lateral views of 3D-reconstructed RB from the sciatic nerve of P60 control and Sema3b ∆SC mice. g Quantification of the RBM indexes from P60 control ( n = 7) and Sema3b ∆SC ( n = 7) mice. h Quantification of the RBM indexes from P7 ( n = 5 for both groups) and P28 Sema3b i∆SC ( n = 5 for both groups) mice with coil (CO) and tamoxifen (TM) treatment at P0. i Measurements of sensitivity to laser heat in control ( n = 6 for all groups) and Sema3b ∆SC ( n = 7 for all groups) mice. j Measurements of sensitivity to von Frey filaments in control ( n = 6) and Sema3b ∆SC ( n = 6) mice. All data are presented as mean ± SEM. * *p < 0.01, * p < 0.05, ns not significant. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a Schematic illustration of the strategy for generating Sema3b tgSC mice. b , c FISH on the sciatic nerve showing an increased Sema3b expression level in SOX10 + SCs of P14 Sema3b tgSC mice. Arrowheads and arrows in b indicate Sema3b + SOX10 + SCs and Sema3b - SOX10 + SCs. Note that in Sema3b tgSC mice, Sema3b expression is ubiquitous in all SOX10 + SCs, which concurrently express TOM. d Quantification of Sema3b transcript level with qPCR in the sciatic nerve of P14 control ( n = 6) and Sema3b tgSC ( n = 6) mice. e , f Representative TEM images of the sciatic nerve from P7 and P28 control and Sema3b tgSC mice. Asterisks and circles denote axons that are adjacent and partially wrapped, respectively. Arrowheads indicate lamellipodia protruding into the endoneurial space. g Quantification of the RBM indexes from P7 and P28 control ( n = 6 for both P7 and P28) and Sema3b tgSC ( n = 6 for both P7 and P28) mice. All data are presented as mean ± SEM. * p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP plot illustrating cell subtype diversity from P14 DRG. b Dot plot comparing expression levels of axon guidance pathway genes in Nefh − nmSNs and Nefh + mSNs. c UMAP plots illustrating the expression levels of Nefh , Nrp1 , Nrp2 , and L1cam in SNs. d–g Transverse sections of P14 sciatic nerve. NRP1 is expressed in TH + ( d ) and CGRP + ( e ) unmyelinated axons, and L1CAM is expressed in almost all unmyelinated axons ( f ). NRP1 and L1CAM are simultaneously expressed in subsets of unmyelinated axons ( g ). The experiment was repeated 3 times on 3 mice. h–k TEM images of the sciatic nerve from P60 control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN mice. l Quantification of the RBM indexes from control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN mice. n = 6 for all groups. m STED images illustrating the distribution of NRP1 and L1CAM particles within cultured sensory axon shafts following Fc and SEMA3B treatment. Arrowheads indicate particles with NRP1 and L1CAM co-localization. n Quantification of the number of L1CAM particles in the axon shaft with Fc ( n = 7 axon shafts from 3 DRGs) and SEMA3B ( n = 8 axon shafts from 3 DRGs) treatment. o Quantification of the numbers of particles with NRP1 and L1CAM co-localization in the axon shaft with Fc ( n = 7 axon shafts from 3 DRGs) and SEMA3B ( n = 8 axon shafts from 3 DRGs) treatment. p Representative images of the sensory axon shafts from control, Nrp1 ∆SN , L1cam ∆SN , and Nrp1/L1cam ∆SN embryos showing the lateral filopodia of axon shaft. q Quantification of the percentages of collapsed sensory growth cones following Fc ( n = 5 DRGs from 3 control embryos) and SEMA3B (control, n = 6 DRGs/3 embryos; Nrp1 ∆SN , n = 6 DRGs/3 embryos; L1cam ∆SN , n = 5 DRGs/3 embryos; Nrp1/L1cam ∆SN , n = 6 DRGs/3 embryos) treatment. r Quantification of the filopodia numbers of cultured DRG shafts following treatment with Fc ( n = 7 axon shafts/3 DRGs) and SEMA3B (control, n = 5 axon shafts/3 DRGs; Nrp1 ∆SN n = 4 axon shafts/3 DRGs; L1cam ∆SN , n = 6 axon shafts/3 DRGs; Nrp1/L1cam ∆SN , n = 4 axon shafts/3 DRGs) treatment. All data are presented as mean ± SEM. *p < 0.05, ** p < 0.01. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Control, Cell Culture

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a STED images showing TUJ1 signals on axon bundles treated with Fc and SEMA3B. Arrowheads indicate axonal beading. b Fasciculation index is calculated as the axonal area divided by the total area. c Quantification of the fasciculation indexes for axon bundles treated with Fc and SEMA3B. n = 6 axon shafts from 3 DRGs for both groups. d KEGG pathway analysis of up-regulated DEGs in nmSN. Pathway enrichment was analyzed by a two-tailed Wald test with Benjamini-Hochberg correction (FDR-adjusted p-value < 0.05 and |log₂ fold change|> 0.25). e Dot plot illustrating genes in the cell adhesion molecules pathway. f , g STED images of P7 Plp1 CreERT2 ;R26 tdTom RBs. Dotted lines demarcate RB boundaries. Asterisks denote axons ensheathed by TOM + mSCs. The experiment was repeated 3 times on 3 mice. f’ , g’ Magnified views of the boxed areas in ( f ) and ( g ). Dotted lines demarcate unmyelinated axon boundaries. Arrowheads indicate lamellipodia undergoing separating fasciculated axons. The asterisk in ( g’ ) denotes an axon wrapped by SC lamellipodia, with strong CDH2 signals in the contact region. h STED images showing TUJ1 signals on axon bundles. i Quantification of the fasciculation indexes for axon bundles with SEMA3B or SEMA3B+Dynasore treatment. n = 6 axons bundles from 3 DRGs for both groups. j , k L1CAM and CDH2 particles on the membrane of sensory axon shafts from E14.5 DRG. l Quantification of artificial fluorescence intensity of L1CAM on the membrane of sensory axon shafts. n = 7 axon bundles from 3 DRGs for all groups. m Quantification of artificial fluorescence intensity of CDH2 on the membrane of sensory axon shafts. n = 6 axon bundles from 3 DRGs for all groups. n–p STED images of the RBs from P7 control, Sema3b tgSC , and Sema3b ∆SC mice. Dotted lines delineate the assumed RB boundaries. q Quantification of the numbers of L1CAM particles per RB. n = 6 RBs from 3 mice for all groups. r Schematic summary: nmSC-derived SEMA3B induces the endocytosis of axonal CAMs, leading to axonal segregation. All data are presented as mean ± SEM. ** p < 0.01, *p < 0.05. Detailed statistics are p rovided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Two Tailed Test, Membrane, Fluorescence, Control, Derivative Assay

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP plot illustrating L1cam expression in iSCs and nmSCs. b FISH on transverse section of P28 sciatic nerve. Arrowheads indicate L1cam + Scn7a + SOX10 + nmSCs and arrows indicate L1cam - Scn7a - SOX10 + mSCs. The experiment was repeated 3 times on 3 mice. c STED image of the RB from P28 Plp1 CreERT2 ;R26 tdTOM sciatic nerves. The experiment was repeated 3 times on 3 mice. c’ Magnified view of the boxed area in ( c ) illustrating a high L1CAM expression level in the contacting area between TUJ1 + axons and TOM + SC lamellipodia. Note the presence of L1CAM signals in TOM + SC lamellipodia. d Drawing a line to calculate the fluorescence intensity across the section. e Quantification of the fluorescence intensities of TOM, TUJ1, and L1CAM signals. f Representative E14.5 DRG SNs cultured on Fc- and L1CAM-coated coverslips. g Quantification of the numbers of attached sensory neurons. n = 6 independent experiments for both group. h SCs purified from P0 sciatic nerves cultured on Fc- or L1CAM-coated coverslips. i Quantification of the numbers of attached SCs. n = 6 independent experiments for both groups. j Assay for mimicking the acute and chronic effect of SEMA3B on attached SNs and SCs on L1CAM-coated coverslip. k Images showing attached SNs under different conditions. l Quantification of the numbers of attached SNs under different conditions. n = 6 independent experiments for all groups. m Images showing attached SCs under different conditions. n Quantification of the numbers of attached SCs under different conditions. n = 6 independent experiments for all groups. o Schematic representation of the three biological events involved in RB assembly: nmA-nmA adhesion, nmA-nmA deadhesion, and nmA-nmSC adhesion. The corresponding molecular mechanisms are depicted. nmSC-derived SEMA3B interacts with axonal NRP1/L1CAM receptor complex to shift CAM-induced nmA-nmA adhesion to CAM-induced nmA-nmSC adhesion. The lower panel presents STED images illustrating the dynamic distribution patterns of CAM during the three events of RB assembly. All data are presented as mean ± SEM. ** p < 0.01, *p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Fluorescence, Cell Culture, Purification, Derivative Assay

Journal: Nature Communications

Article Title: SEMA3B switches axon-axon to axon-glia interactions required for unmyelinated axon envelopment and integrity

doi: 10.1038/s41467-025-61318-8

Figure Lengend Snippet: a UMAP visualization of SCs from scRNA-seq data in sham mice and nerve-injury mice. b Relative proportions of SC lineages in total SCs of sham mice and nerve-injury mice. c Dot plot showing expression levels of Scn7a , Sema3b , and L1cam in SCs of sham mice and nerve-injury mice. d Cartoon depicting that the distal part of the injury site is harvested for analysis. e Sema3b expression levels in SOX10 + SC following different days post ligation (dpl) of control mice. f Sema3b expression levels in SOX10 + SCs following different days post-ligation (dpl) of Sema3b tgSC mice. Sema3b transcript levels demonstrated relatively stable after nerve injury. g Quantification using qPCR showing Sema3b transcript levels in sciatic nerves without injury (sham) and with 7 and 21 days post ligation (dpl). n = 6 mice for all groups. h–j Representative TEM images of the sciatic nerves from control and Sema3b tgSC mice. Note that the contact area between nmSC lamellipodia and axons is significantly decreased in the RB of control mice after injury. In contrast, the deficient RB assembly is noticeably reduced in Sema3b tgSC mice with injury. Asterisks and circles indicate axons that are adjacent and partially wrapped, respectively. Arrowheads indicate lamellipodia protruding into the endoneurial space. k Quantification of RBM indexes of control and Sema3b tgSC mice without and with nerve crush. n = 6 mice for all groups. l Measurements of sensitivity to von Frey filaments in control and Sema3b tgSC mice without and with nerve injury. n = 6 mice for all groups . m Measurements of sensitivity to hot laser in control and Sema3b tgSC mice without and with nerve injury. n = 6 mice for all groups. All data are presented as mean ± SEM. * *p < 0.01, *p < 0.05. Detailed statistics are provided in Supplementary Table . Source data are provided as a file.

Article Snippet: After a 24-h culture period, SEMA3B (R&D Systems, 5440-S3-025/CF), with or without Dynasore, was introduced into the medium for a 2-h incubation duration.

Techniques: Expressing, Ligation, Control

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 2. (A) Effects of zaltoprofen on the viability of OUMS27 and SW1353 cells. The cells were treated with or without zaltoprofen for 72 h, followed by WST assay. Asterisks denote a statistically significant difference (*P < 0.05) between cells with and without treatment of zaltoprofen (200 or 400 μmol/L). (B) Representative photos of 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay in PPARγ knockdown cell lines, OUMS27shPPAR and SW1353shPPAR, and the control cell lines, OUMS27shNTC and SW1353shNTC with or without the treatment of zaltoprofen for 24 h. Scale bar = 100 μm. (C, D) The EdU-positive cell ratio [EdU-positive nuclei (red)/Hoechst 33342-positive nuclei (blue)] in OUMS27 (C) and SW1353 cells (D). Values represented as the mean ± SEM. Asterisks denote a statistically significant difference (*P < 0.05 and **P < 0.01).

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: WST Assay, Proliferation Assay, Knockdown, Control

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 1. (A) Effects of zaltoprofen on PPARγ mRNA expression in OUMS27 and SW1353 cells by qRT-PCR. Values represented as the mean ± SEM. Asterisks denote a statistically significant difference (*P < 0.05 and **P < 0.01) between cells with and without treatment of zaltoprofen (400 μmol/L). (B) Effects of zaltoprofen on PPARγ protein expression in OUMS27 and SW1353 cells by western blotting. GAPDH was used as an internal control. (C) PPARγ activity was determined by luciferase reporter gene assay. Reporter cells were treated with zaltoprofen (0–200 μmol/L) for 24 h, followed by measurement of luciferase activities.

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 3. (A) Cell migration assay was carried out using PPARγ knockdown cell lines, OUMS27shPPAR and SW1353shPPAR, and the control cell lines, OUMS27shNTC and SW1353shNTC with or without the treatment of zaltoprofen. A selective inhibitor of MMP2, ARP100, was used at 25 μmol/L. Scale bar = 100 μm. (B, C) The fielded wound area (%) was calculated in OUMS27 (B) and SW1353 cells (C). Values represented as the mean ± SEM. Asterisks denote a statistically significant difference (*P < 0.05 and **P < 0.01).

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Cell Migration Assay, Knockdown, Control

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 4. (A) Cell invasion assay was carried out using a PPARγ knockdown cell line SW1353shPPAR and the control cell line SW1353shNTC with or without the treatment of zaltoprofen. A selective inhibitor of MMP2, ARP100, was used at 25 μmol/L. Scale bar = 100 μm. (B) Relative invaded cell number was calculated. Values represented as the mean ± SEM. Asterisks denote a statistically significant difference (**P < 0.01). (C) Gelatin zymography. Effects of zaltoprofen (400 μmol/L) on MMP2 activities in PPARγ knockdown cell lines, OUMS27shPPAR and SW1353shPPAR, and the control cell lines, OUMS27shNTC and SW1353shNTC.

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Invasion Assay, Knockdown, Control, Zymography

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 5. (A, B) Sagittal, MRI T2 weighted image of cervical spine at first visit on the initial hospital before (A) and after (B) the surgery. The tumor (arrow) was resected (1st operation), which markedly compressed the spinal cord. (C) Local recurrence was detected at C5-6 and tumor excision (2nd operation) was then performed after 1 year from the initial diagnosis. An arrow indicates the tumor. (D) The residual tumor (arrow). (E) Sagittal, MRI T2 fat suppression image. The tumors markedly enlarged and severe pain occurred in his left hand. The tumor of C2-4 was resected, while the tumor of C5-6 was unresectable. Zaltoprofen (240 mg/day) was then administrated for the pain. (F) Before the final operation for severe cervical kyphosis, MRI T2 fat suppression image showed the localized tumor of C5-C6 remained.

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Biomarker Discovery

Journal: Cancer medicine

Article Title: Anti-tumor effects of a nonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator-activated receptor gamma and suppressing matrix metalloproteinase-2 expression.

doi: 10.1002/cam4.1438

Figure Lengend Snippet: Figure 6. Histopathological findings of the tumors at the second operation (preadministration of zaltoprofen, Pre) and the final operation (postadministration of zaltoprofen, Post). Scale bar = 25 μm. (A) HE staining. (B, C) Immunofluorescent stainings for PPARγ (B) and MMP2 (C). DAPI, a nuclear staining.

Article Snippet: Zaltoprofen and ARP100, a selective MMP2 inhibitor, were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Staining

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-c , NINJ1 oligomerization (Native-PAGE) and LDH-release from WT and NINJ1 KO iPSDMs pretreated with glycine or inhibitors as indicated and infected with Mtb mc²6206, MOI 20 for 4h ( a, b ), Mtb H37Rv, 7.5 MOI for 24h ( c ), or treated with cell death stimuli for 4h: d , pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950; e , apoptosis – venetoclax; f , necroptosis – Z-VAD-FMK+BV-6+TNFa (zBT) w/wo RIPK3 inhibitor GSK’872; g , ferroptosis – RSL-3 w/wo Ferrostatin-1. Data are means (bars) ± s.e.m. of at least three independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test of treatments within each cell line and stimuli between cell lines. For gel source data, see Supplementary Figure 1.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Clear Native PAGE, Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, Lysates of WT, NINJ1 KO and GSDMD KO iPSC-derived monocytes (m) and macrophages (M) were subjected to SDS-PAGE and immunoblotted for NINJ1, GSDMD and β-actin. A3 and C6 are NINJ1 KO clones; A7, B8 are GSDMD KO clones. b, Human monocyte-derived macrophages were transfected with siRNA targeting NINJ1 or a non-targeted control and infected with Mtb mc²6206, MOI 20, for 4h. Shown is LDH-release and NINJ1 expression level by qPCR. Data are presented as mean ± s.e.m of four donors (shown with individual symbols). *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with uncorrected Fisher’s LSD. c-j, LDH-release from iPSDM WTs and NINJ1 KO clones A3, C6 ( c-f ) or GSDMD KO clones A7, B8 ( g-j ) pretreated with glycine or inhibitors as indicated and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( c, g ); apoptosis – venetoclax ( d, h ); necroptosis – Z-VAD-FMK+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( e, i ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( f, j ). ( c-f ) Data are means (bars) ± s.e.m. of three (A3) or two (C6) independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test. ( g-j ) Data are means (bars) ± s.d. of two or three technical replicates (circles) from 1 independent experiment.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Derivative Assay, SDS Page, Clone Assay, Transfection, Control, Infection, Expressing

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-d , Cytokine release from WT iPSDMs pretreated with glycine or inhibitors as indicated for 30 min and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( a ); apoptosis – venetoclax ( b ); necroptosis – zVAD+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( c ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( d ). Cytokine levels were assessed by multiplex ELISA. Data are means (bars) of two individual experiments (circles), each with two replicates.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a , Representative images of NINJ1 KO THP-1 cells expressing hNINJ1-mNG (THP1-NINJ1-mNeonGreen) pretreated with glycine ( a , right column) before cell death stimuli venetoclax (apoptosis), LPS + nigericin (LPS+N, pyroptosis), RSL-3 (ferroptosis) for 4h, or infection with Mtb mc26206-BFP for 20h. Time-lapse TIRF (mNG), widefield (DRAQ7 and BFP) and brightfield microscopy images of NINJ1-mNG (green), DRAQ7 (red) and Mtb (blue), and morphology, respectively. Scale bars 10 mm. b , Quantification of NINJ1-mNG puncta densities at 4h (LPS+N, RSL-3) or 20h (Mtb) as represented in ( a ). Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c , Representative time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 uptake (red) upon Mtb mc26206-BFP infection (blue) temporally aligned by start of NINJ1 clustering (t=0). Time in min. Scale bars 10 mm. d, e , Fitted curves ( d ) and t50 values (x value where y=50% max.) of sigmoidal fits ( e ) of NINJ1 oligomerization kinetics during Mtb-infection, apoptosis (venetoclax), pyroptosis (LPS+N) or ferroptosis (RSL-3) monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) or 5 min (Mtb-infection). 3-5 cells analyzed per condition. Individual kinetic curves and representative images for RCD induction are shown in Extended Data Figure 3.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Expressing, Infection, Microscopy

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Quantification of NINJ1-mNG puncta densities monitored by TIRF microscopy ( a ) and phagocytosed bacteria monitored by intracellular BFP intensities ( b ) before NINJ1 inhibition by glycine and after 4h of RCD induction (Venetoclax, LPS+N, RSL-3) or after 3h and 20h of Mtb mc²6206-BFP infection. Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c,d, Kinetics of NINJ1 puncta densities and DRAQ7 intensities monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) with (c) representative Time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 (WF, red) aligned by start of NINJ1 clustering. Time in sec. Scale bars 10 μm. Intensity values were normalized to the highest value for each cell and aligned by the start of NINJ1 oligomerization. Data are means ± s.d. for 3-5 cells per condition with sigmoidal fits for NINJ1-mNG puncta density increase (green) and mono-exponential increase for DRAQ7 intensities (red).

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Microscopy, Bacteria, Inhibition, Infection

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial NDUFA4L2 attenuates the apoptosis of nucleus pulposus cells induced by oxidative stress via the inhibition of mitophagy

doi: 10.1038/s12276-019-0331-2

Figure Lengend Snippet: After pretreatment with FCCP or CsA, primary nucleus pulposus cells were exposed to 400 μM TBHP for 6 h. a Western blotting for the protein levels of cleaved caspase-3, Bcl-2/Bax, Beclin-1, Parkin and LC-3II. b–f Quantitative analysis of the protein content of cleaved caspase-3, Bcl-2/Bax, Beclin-1, Parkin and LC-3II. g Flow cytometry was used to detect apoptosis in nucleus pulposus cells. h Quantitative analysis of flow cytometry for the detection of apoptosis. i Fluorescence images of NP cells infected with mRFP-GFP-LC-3 adenovirus. sensGFP is sensitive to the pH changes owing to the fusion of autophagosomes and lysosomes, whereas mRFP is stable. When autophagy was induced, autophagosomes and lysosomes were fused, sensGFP was quenched and mRFP was increased. j Hoechst 33258 staining detected apoptosis in the cell nucleus. Nuclear condensation was observed in apoptotic cells. The data are presented as the mean ± S.D. *** p < 0.001, ** p < 0.01, * p < 0.05 ( n = 5). Cd caspase-3: cleaved caspase-3.

Article Snippet: The membranes were blocked with TBST-buffered saline solution containing 5% dry milk for 2 h and then incubated overnight at 4 °C or at 25 °C for 2 h with primary antibodies against HIF-1α (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Beclin-1 (1:1000, CST, USA), LC-3 (1:1000, Novus, USA), cleaved caspase-3 (1:1000, Proteintech, China), cleaved PARP (1:1000, CST, USA), NDUFA4L2 (1:1000, Proteintech, China), and P62 (1:1000, Proteintech, China).

Techniques: Western Blot, Flow Cytometry, Fluorescence, Infection, Staining

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial NDUFA4L2 attenuates the apoptosis of nucleus pulposus cells induced by oxidative stress via the inhibition of mitophagy

doi: 10.1038/s12276-019-0331-2

Figure Lengend Snippet: Primary nucleus pulposus cells were transfected with si-NDUFA4L2, si-NC, pc-NDUFA4L2 or pc-NC for 48 h and then exposed to TBHP. a Western blotting for the protein levels of NDUFA4L2, LC-3II and Parkin was performed to determine the function of si-NDUFA4L2. b Western blotting for the protein levels of NDUFA4L2, LC-3II and Parkin was performed to determine the function of pc-NDUFA4L2. c Western blotting for the protein levels of NDUFA4L2, LC-3II, Parkin and Bcl-2/Bax was performed to investigate the function of NDUFA4L2 in NP cells exposed to TBHP. d–f Quantitative analysis of the protein content of P62, Bcl-2/Bax and LC-3II. g Immunofluorescence images showing staining for NDUFA4L2 (red), cytochrome C (green), and DAPI (blue) and merged images of two signals. h Immunofluorescence images showing staining for NDUFA4L2 (red), LC-3 (green), and DAPI (blue) and merged images of two signals. The data are presented as the mean ± S.D. *** p < 0.001, ** p < 0.01, * p < 0.05 ( n = 5).

Article Snippet: The membranes were blocked with TBST-buffered saline solution containing 5% dry milk for 2 h and then incubated overnight at 4 °C or at 25 °C for 2 h with primary antibodies against HIF-1α (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Beclin-1 (1:1000, CST, USA), LC-3 (1:1000, Novus, USA), cleaved caspase-3 (1:1000, Proteintech, China), cleaved PARP (1:1000, CST, USA), NDUFA4L2 (1:1000, Proteintech, China), and P62 (1:1000, Proteintech, China).

Techniques: Transfection, Western Blot, Immunofluorescence, Staining

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial NDUFA4L2 attenuates the apoptosis of nucleus pulposus cells induced by oxidative stress via the inhibition of mitophagy

doi: 10.1038/s12276-019-0331-2

Figure Lengend Snippet: Primary nucleus pulposus cells were transfected with si-NDUFA4L2 or si-NC for 48 h and then exposed to FCCP or CsA. a Western blotting for the protein levels of NDUFA4L2, cleaved caspase-3, P62, Parkin and LC-3II. b – f Quantitative analysis of the protein content of NDUFA4L2, cleaved caspase-3, P62, Parkin and LC-3II. g Fluorescence images of NP cells infected with mRFP-GFP-LC-3 adenovirus. sensGFP is sensitive to the pH changes due to the fusion of autophagosomes and lysosomes, whereas mRFP is stable. When autophagy was induced, autophagosomes and lysosomes were fused, sensGFP was quenched and mRFP was increased. The data are presented as the mean ± S.D. *** p < 0.001, ** p < 0.01, * p < 0.05 ( n = 5).

Article Snippet: The membranes were blocked with TBST-buffered saline solution containing 5% dry milk for 2 h and then incubated overnight at 4 °C or at 25 °C for 2 h with primary antibodies against HIF-1α (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Beclin-1 (1:1000, CST, USA), LC-3 (1:1000, Novus, USA), cleaved caspase-3 (1:1000, Proteintech, China), cleaved PARP (1:1000, CST, USA), NDUFA4L2 (1:1000, Proteintech, China), and P62 (1:1000, Proteintech, China).

Techniques: Transfection, Western Blot, Fluorescence, Infection