Journal: bioRxiv

Article Title: Extrinsic MMPs drive epithelial shape change via basal ECM disassembly in the Drosophila wing disc

doi: 10.64898/2026.01.21.700823

Figure Lengend Snippet: (A–B’’) Wing discs carrying mmp1-GFP transcriptional reporter (green) and expressing RedStinger under the control of 1151-Gal4 (magenta) or btl-Gal4 (cyan) were dissected at 1–1.5 h after puparium formation (APF) and stained with anti-GFP antibody and DAPI (white). Orange arrowheads indicate Mmp1-positive myoblasts or tracheal cells. An asterisk indicates a Mmp1-positive tracheal cell shown in (A). Scale bar, 50 μm. (C–C’’) A wing disc carrying the Mmp2::GFP knock-in reporter (green) and expressing RedStinger under the control of 1151-Gal4 (magenta) at 1–1.5 h APF, stained with anti-GFP antibody and DAPI (white). Orange arrowheads indicate Mmp2-positive myoblasts. Scale bar, 50 μm. (D–D’’) A wing disc carrying the Mmp2::GFP knock-in reporter (green) at 1–1.5 h APF, stained with anti-GFP and anti-Gasp (cyan) antibodies, together with DAPI (white). Orange dotted lines outline the tracheal tube. Orange arrowheads indicate Mmp1-positive tracheal cells. Scale bar, 50 μm. (E–H’) Wing discs carrying viking::GFP (pseudocolor) were dissected, treated with ecdysone, and stained with anti-GFP antibody and DAPI (white). Images are shown for control (E) and for discs expressing Timp under the control of 1151-Gal4 (F), btl-Gal4 (G), or both 1151-Gal4 and btl-Gal4 (H). Yellow dotted lines outline the notum regions. Scale bar, 50 μm. (I) Dot plots showing the percentage of Collagen IV-disassembled area in the notum, where Collagen IV signals could be more reliably quantified than in the hinge region, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (E) (n = 23), (F) (n = 15), (G) (n = 21), and (H) (n = 14). *p<0.05, ****p<0.0001; pairwise comparisons using the Wilcoxon rank-sum test with BH correction. (J–M) Time-lapse confocal imaging of the ecdysone-treated wing discs carrying ubi-RFP (magenta). Images are shown for control (J) and for discs expressing Timp under the control of 1151-Gal4 (K), btl-Gal4 (L), or both 1151-Gal4 and btl-Gal4 (M). Orange arrowheads indicate the same anatomical position before and after the curvature change, marking the region that normally undergoes the concave-to-convex transition. Time is shown as h:min from the onset of ecdysone treatment. Scale bar, 100 μm. (N) Quantification of the final wing disc shape, shown as the percentage of discs that remained concave or transitioned to a convex shape, corresponding to the representative images shown in (J) (n = 44), (K) (n = 32), (L) (n = 35), and (M) (n = 20). *p<0.05, **p<0.01, ****p<0.0001; Fisher’s exact test with Holm correction.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000), rabbit anti-GFP antibody (Invitrogen, #A6455, 1:500), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) antibody (CST, #3671, 1:100), rabbit anti-Zfh1 antibody (R. Lehmann, 1:1000), rabbit anti-DsRed antibody (Clontech, # 632496, 1:500), mouse anti-Gasp antibody (DSHB, #2A12, 1:100), anti-Mmp1 antibody (DSHB, 1:10 from 1:1:1 cocktail of 3A6B4, 3B8D12 and 5H7B11) (PMID: 25224221), and Rabbit anti-Collagen IV antibody (1:100) (PMID: 40570847).

Techniques: Expressing, Control, Staining, Knock-In, Imaging

Journal: bioRxiv

Article Title: Extrinsic MMPs drive epithelial shape change via basal ECM disassembly in the Drosophila wing disc

doi: 10.64898/2026.01.21.700823

Figure Lengend Snippet: (A–D’’) Wing discs were dissected, treated with ecdysone, and stained with anti-Mmp1 (green) and anti-Zfh1 (magenta) antibodies. Images are shown for discs expressing empty-RNAi (VDRC60100) (A–B’) or EcR-RNAi (NIG1765R-2) (C–D’), together with Dicer2 (Dcr2), under the control of 1151-Gal4 and btl-Gal4 . (B–B’) and (D–D’) show magnified views of the regions outlined by cyan dotted lines in (A–A’) and (C–C’), respectively. Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Orange arrowheads indicate the anti-Mmp1-positive puncta surrounding Zfh1-positive myoblast nuclei. Scale bar, 10 μm. (E) Dot plots showing the mean number of anti-Mmp1-positive puncta per myoblast, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (A–B) (n = 10) and (C–D) (n = 9). ****p<0.0001; Wilcoxon rank-sum test. (F–G’) Wing discs carrying Mmp2::GFP knock-in reporter (pseudocolor) were dissected, treated with ecdysone, and stained with anti-GFP and anti-Zfh1 (magenta) antibodies. Images are shown for discs expressing yellow-RNAi (NIG3757R-1) (F–F’) or EcR-RNAi (NIG1765R-2) (G–G’), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae bearing Tub-Gal80 ts were maintained under the temperature conditions indicated in Fig. S5A. Scale bar, 50 μm. (H) Dot plots showing the mean intensity of Mmp2::GFP knock-in reporter in myoblasts, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (F) (n = 8) and (G) (n = 11). *p<0.05; Wilcoxon rank-sum test. (I–J’) Wing discs were dissected, treated with ecdysone, and stained with anti-Collagen IV antibody (pseudocolor) and DAPI (white). Images are shown for discs expressing empty-RNAi (VDRC60100) (I) or EcR-RNAi (NIG1765R-2) (J), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Scale bar, 50 μm. (K) Dot plots showing the percentage of the Collagen IV-disassembled area in the notum, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (I) (n = 17) and (J) (n = 17). **p<0.01; Wilcoxon rank-sum test. (L–N’) Wing discs were dissected and treated with ecdysone for 24 h. Images are shown for discs expressing empty-RNAi (VDRC60100) (L), EcR-RNAi (NIG1765R-2) (M), or EcR-RNAi (BDSC9327) (N), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Schematic diagrams (L’, M’ and N’) illustrate the concave and convex shapes corresponding to the representative images shown in (L), (M), and (N). (O) Quantification of the final wing disc shape, shown as the percentage of discs that remained concave or transitioned to a convex shape, corresponding to the representative images shown in (L) (n = 79), (M) (n = 48), and (N) (n =53). *p<0.05; Fisher’s exact test with Holm correction.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000), rabbit anti-GFP antibody (Invitrogen, #A6455, 1:500), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) antibody (CST, #3671, 1:100), rabbit anti-Zfh1 antibody (R. Lehmann, 1:1000), rabbit anti-DsRed antibody (Clontech, # 632496, 1:500), mouse anti-Gasp antibody (DSHB, #2A12, 1:100), anti-Mmp1 antibody (DSHB, 1:10 from 1:1:1 cocktail of 3A6B4, 3B8D12 and 5H7B11) (PMID: 25224221), and Rabbit anti-Collagen IV antibody (1:100) (PMID: 40570847).

Techniques: Staining, Expressing, Control, Knock-In

Journal: International Journal of Molecular Medicine

Article Title: Targeting of CIRP attenuates osteoarthritis progression via suppressing TLR4/NF-κB/NLRP3 signaling axis

doi: 10.3892/ijmm.2025.5674

Figure Lengend Snippet: CIRP expression in chondrocytes from healthy individuals and OA patients. (A) Trypan blue staining of chondrocytes. (B) Representative quantitative PCR showing the expression of CIRP, collagen Ⅱ, MMP1, MMP3, MMP13 and ADAMTS5 in chondrocytes from healthy individuals and OA patients. (C and D) Western blotting showing the expression of CIRP, collagen Ⅱ, MMP1, MMP3, MMP13 and ADAMTS5 in chondrocytes from healthy individuals and OA patients. (n=5), * P<0.05, ** P<0.01, *** P<0.001. CIRP, cold-inducible RNA-binding protein; OA, osteoarthritis; MMPs, matrix metalloproteinases; ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5.

Article Snippet: Following a transfer at 100 V for 1 h at 4°C, the membrane was blocked with 5% skimmed milk for 1 h at room temperature before being incubated with primary antibodies against GADPH (1:50,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.), MMP1 (1:1,000, 10371-2-AP, ProteinTech Group, Inc.), MMP3 (1:5,000; cat. no. 66338-1-Ig; ProteinTech Group, Inc.), MMP13 (1:1,000; cat. no. 18165-1-AP; ProteinTech Group, Inc.), a disintegrin and metalloproteinase with thrombospondin motif-5 (Adamts5; 1:500; cat. no. A2836; ABclonal Biotech Co., Ltd.), iNOS (1:500; cat. no. 18985-1-AP; ProteinTech Group, Inc.), cyclooxygenase-2 (COX-2; 1:500; cat. no. 12375-1-AP; ProteinTech Group, Inc.), lamin B1 (1:5,000; cat. no. 12987-1-AP; ProteinTech Group, Inc.), CD63 (1:500; cat. no. 32151-1-AP, ProteinTech Group, Inc.), CD9 (1:500; cat. no. 84801-13-RR, ProteinTech Group, Inc.), GM130 (1:500; cat. no. 11308-1-AP, ProteinTech Group, Inc.), Human Reactive Inflammasome Antibody Sampler Kit II (1:1,000; cat. no. 25620T; Cell Signaling Technology Inc.), CIRP (1:1,000; cat. no. 68522; Cell Signaling Technology Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology Inc.), IκBα (1:1,000, cat. no. 4814; Cell Signaling Technology Inc.), and collagen II (1:500; cat. no. AF6528, Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot, RNA Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Targeting of CIRP attenuates osteoarthritis progression via suppressing TLR4/NF-κB/NLRP3 signaling axis

doi: 10.3892/ijmm.2025.5674

Figure Lengend Snippet: CIRP promotes inflammatory response and ECM degradation in human chondrocytes. (A) Quantitative PCR analysis showing the effect of CIRP on the expression of inflammatory cytokines, including IL-6, IL-1β, TNF-α, iNOS and COX-2. (B) Western blotting showing the protein expression of iNOS and COX-2. (C and D) Quantitative PCR and western blotting showing the effect of CIRP on the expression of MMP1, MMP3, MMP13, ADAMTS5 and collagen Ⅱ. (E) Immunofluorescence analysis of collagen Ⅱ and MMP13 expression following CIRP treatment, with DAPI staining for nuclei (scale bar, 50 μ m). n=3, * P<0.05, ** P<0.01, *** P<0.001. CIRP, cold-inducible RNA-binding protein; ECM, extracellular matrix; iNOS, nitric oxide synthase; COX-2, cyclooxygenase-2; MMPs, matrix metalloproteinases; ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5.

Article Snippet: Following a transfer at 100 V for 1 h at 4°C, the membrane was blocked with 5% skimmed milk for 1 h at room temperature before being incubated with primary antibodies against GADPH (1:50,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.), MMP1 (1:1,000, 10371-2-AP, ProteinTech Group, Inc.), MMP3 (1:5,000; cat. no. 66338-1-Ig; ProteinTech Group, Inc.), MMP13 (1:1,000; cat. no. 18165-1-AP; ProteinTech Group, Inc.), a disintegrin and metalloproteinase with thrombospondin motif-5 (Adamts5; 1:500; cat. no. A2836; ABclonal Biotech Co., Ltd.), iNOS (1:500; cat. no. 18985-1-AP; ProteinTech Group, Inc.), cyclooxygenase-2 (COX-2; 1:500; cat. no. 12375-1-AP; ProteinTech Group, Inc.), lamin B1 (1:5,000; cat. no. 12987-1-AP; ProteinTech Group, Inc.), CD63 (1:500; cat. no. 32151-1-AP, ProteinTech Group, Inc.), CD9 (1:500; cat. no. 84801-13-RR, ProteinTech Group, Inc.), GM130 (1:500; cat. no. 11308-1-AP, ProteinTech Group, Inc.), Human Reactive Inflammasome Antibody Sampler Kit II (1:1,000; cat. no. 25620T; Cell Signaling Technology Inc.), CIRP (1:1,000; cat. no. 68522; Cell Signaling Technology Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology Inc.), IκBα (1:1,000, cat. no. 4814; Cell Signaling Technology Inc.), and collagen II (1:500; cat. no. AF6528, Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, RNA Binding Assay