Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a , b Immunofluorescence staining and quantification of phalloidin, γ-H2AX and L1-CT 48 h post IR (2 Gy, 5 Gy, or 10 Gy) in HUVECs (left panels, magnification, ×400). Scale bar = 20 µm (enlarged, 5 µm). Bar graphs quantifying the number of γ-H2AX foci, the phalloidin density, and the number of colocalized foci (right panels). For quantification of phalloidin density, error bars represent mean ± SEM (2 Gy vs. 10 Gy p < 0.0001; 5 Gy vs. 10 Gy p = 0.0015). For quantification of the number of γ-H2AX foci, error bars represent mean ± SD (**** p < 0.0001). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (bottom panels). Error bars represented mean ± SEM (**** p < 0.0001). c Heat ma p of the RNA-seq analysis results showing radiation-induced EndMT and the mesenchymal phenotype. Total RNA was isolated from HUVECs before and after 10 Gy IR (5 h, 72 h). d Immunofluorescence staining of L1-CT and γ-H2AX in HUVECs at 0, 1, 24, and 48 h post IR (10 Gy; left panels). Quantification of γ-H2AX foci and nuclear L1CAM (magnification, ×400; right panels). The number of γ-H2AX foci with an intensity greater than 40 and a foci diameter of 0.1 μm was counted. Scale bar = 20 µm (enlarged, 5 µm). e Immunoblotting and quantification of full-length L1CAM and L1-CT fragments from HUVECs transfected with lentiviral shRNA targeting L1CAM 48 h after IR (10 Gy; upper panels). Error bars represent mean ± SD (full-length L1CAM: sh-Control IR – vs. + p = 0.0003; sh-Control IR + vs. sh-L1CAM IR + p < 0.0001, L1-CT fragments: sh-Control IR – vs. + p = 0.0013; sh-Control IR + vs. sh-L1CAM IR + p = 0.0006). Immunofluorescence staining of γ-H2AX and L1-CT 48 h post IR (10 Gy) in HUVECs (magnification, ×400). Scale bar = 5 µm. f Immunoblotting (upper panel) of full-length L1CAM and L1-CT fragments in the cytoplasmic (C) and nuclear (N) fractions of HUVECs 48 h post IR (10 Gy). Quantification of full-length L1CAM in the cytoplasmic fractions and L1-CT fragments in the nuclear fractions. HUVECs were treated with control IgG or Ab417 (20 µg/mL) before IR. GAPDH and lamin B were used as cytoplasmic and nuclear markers, respectively. Error bars represent mean ± SD from independent experiments (full-length L1CAM: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0001, L1-CT fragments: No IR vs. IR + IgG p = 0.0228; IR + IgG vs. IR + Ab417 p = 0.0448). g Immunofluorescence staining for L1-CT and γ-H2AX 0 and 48 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL) (upper panel). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400) (lower panel). Scale bar = 20 µm (enlarged, 5 µm). h Immunofluorescence staining (upper panel) and quantification (lower panel) of phalloidin and L1-CT 72 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD ( p = 0.0007). i Immunofluorescence staining for L1-CT and γ-H2AX at 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; left panel). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (middle panels). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400; right panel). Scale bar = 5 µm. j Immunofluorescence staining and quantification of phalloidin and γ-H2AX 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (magnification, ×400). Scale bar = 10 μm. Error bars represent mean ± SD (IgG vs. Dox + IgG p = 0.0002; Dox + IgG vs. Dox + Ab417 p = 0.0482; Ab417 vs. Dox + Ab417 p = 0.0065). For quantification of γ-H2AX foci and γ-H2AX foci colocalized with L1CAM, the foci in each sample were counted at least 70 cells per field (magnification, ×100). The average number of foci/cell was determined from >6 fields (magnification, ×100). Data are representative of three independent experiments. ( h : two-talied Student’s t -test, all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Amplification, RNA Sequencing, Isolation, Western Blot, Transfection, shRNA, Control

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a Immunofluorescence staining and quantification of γ-H2AX foci colocalized with L1-CT (upper left panel; magnification, ×400) and immunoblotting (lower left panel) and quantification (right panels) of full-length L1CAM, L1-CT fragments, and γ-H2AX at 48 h post-irradiation (10 Gy) in HUVECs with or without γ-secretase inhibitor L-685,458 (3 µM) treatment. For quantification of γ-H2AX foci colocalized with L1-CT, error bars represent mean ± SEM ( p = 0.0017). For quantification of full-length L1CAM and L1-CT fragments, error bars represent mean ± SD (full-length L1CAM: (-) vs. IR p = 0.0011; IR vs. IR + L-685,458 p = 0.0233, L1-CT fragments: (-) vs. IR p = 0.0017; IR vs. IR + L-685,458 p = 0.0177). b , c HUVECs were transfected with human full-length (L1-WT), NLS-mutated (L1-4A), and endocytosis-deficient (L1-dRSLE) L1CAM vectors after knockdown of endogenous L1CAM. b Scheme of L1-WT, L1-4A and L1-dRSLE constructs (top left panel). Immunoblotting of full-length L1CAM in HUVECs (top right panel) and immunofluorescence staining (middle panels) and quantification (bottom panels) of L1-CT and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SEM (No. γ-H2AX foci: Control vs. L1CAM-WT p = 0.001; L1CAM-WT vs. L1CAM-4A p = 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0002, No. L1-CT foci: **** p < 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0004, No. colocalized foci: Control vs. L1CAM-WT p = 0.006; **** p < 0.0001). c Immunofluorescence staining and quantification of phalloidin and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD (Control vs. L1CAM-WT p = 0.0096; L1CAM-WT vs. L1CAM-4A p = 0.0014; **** p < 0.0001). d Immunofluorescence staining and pearson’s correlation coefficient of L1-CT colocalized with p-ATM, 53BP1, and DNA-PKcs in the nuclei of Ab417-pre-treated HUVECs 48 h post-irradiation (10 Gy magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SD (* * ** p < 0.0001). e Flow cytometry analysis of cells with GFP positivity resulting from DNA repair and quantification of HR (upper panel) and NHEJ (lower panel) efficiency in L1CAM-knockdown HUVECs. The HUVECs were transiently transfected with the DR-GFP or EJ5-GFP construct along with control or L1CAM siRNA and were then transfected with a SceI plasmid to induce DNA damage. Error bars represent mean ± SD (HR efficacy p = 0.0005, NHEJ efficacy p = 0.0011). f Immunofluorescence staining (upper panel) for GFP, phalloidin, γ-H2AX in HUVECs 48 h post-irradiation (10 Gy). Quantification of phalloidin is shown (magnification, ×400; right panel). Scale bar = 20 µm. Error bars represent mean ± SD (No IR vs. IR + Control p = 0.0001; IR + Control vs. IR + L1CAM-WT p = 0.0063; IR + L1CAM-WT vs. IR + L1CAM-C-term p = 0.0027, IR + Control vs. IR + L1CAM-C-term p < 0.0001). HUVECs were transfected with L1-WT or L1-CT lentiviral vectors tagged with N-terminal GFP and C-terminal His. Transfection efficiency (left panel) was tested by immunoblotting for L1-WT and L1-CT. For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in a minimum of 70 cells per field (magnification, ×100). The average number of colocalized foci/cell was determined from five fields (magnification, ×100). The data are presented the means ± SDs and ±SEMs from three independent experiments. ( a upper left panel: two-talied Student’s t -test; a all other panels and b , c , f : one-way ANOVA for multiple comparisons; d , e two-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Western Blot, Irradiation, Transfection, Knockdown, Construct, Control, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a RT-qPCR analysis of L1CAM in p53-knockdown HUVECs 48 h post-irradiation (10 Gy). Error bars represent mean ± SD from three independent experiments ( p < 0.0001). b Immunoblotting (upper panel) and quantification (lower panel) of full-length L1CAM and L1-CT 48 h post-irradiation (10 Gy) in p53-knockdown HUVECs pre-treated with control IgG or Ab417 (20 µg/mL). Error bars represent mean ± SD from four independent experiments (full-length L1CAM: No IR vs. si-Control IR + IgG p = 0.0468; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0468; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0462; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0218, L1-CT: No IR vs. si-Control IR + IgG p = 0.0124; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0484; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0206; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0004). c Immunofluorescence staining (upper panel) and quantification (lower panel) for γ-H2AX and L1-CT. The results of quantification of γ-H2AX foci, nuclear L1CAM, and colocalization of γ-H2AX foci with L1CAM are shown (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (No. γ-H2AX foci: si-Control vs. si-p53+IgG p = 0.0107; si-p53+IgG vs. si-p53 + Ab417 p = 0.0242, No. L1-CT foci: si-Control vs. si-p53+IgG p = 0.0425; si-p53+IgG vs. si-p53 + Ab417 p = 0.0119, No. colocalized foci: si-Control vs. si-p53 + IgG p = 0.0131; si-p53 + IgG vs. si-p53 + Ab417 p = 0.0097). For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in at least 70 cells per field (magnification, ×100, n = 5). d , e Wild-type or EC-p53KO mice were injected intravenously with control IgG or Ab417 (10 mg/kg) and subjected to 17.5 Gy thoracic irradiation ( n = 5 animals per group). d Immunofluorescence staining (upper panels) for γ-H2AX, L1CAM, and CD31 in heart tissues 3 weeks post-irradiation and quantification (lower panels) of γ-H2AX + cells and nuclear L1CAM + cells among CD31 + cells (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (γ-H2AX + in CD31 + nuclei: WT + IR vs. p53KO+IR p = 0.0005; p 53KO + IgG vs. p53KO + Ab417 p < 0.0001, L1CAM + in CD31 + nuclei: WT + IR vs. p53KO+IR p = 0.0001; p53KO+IgG vs. p 53KO + Ab417 p = 0.0002). e Haematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical detection of CD31 in heart tissues 3 weeks post-irradiation (upper panels) and quantification of ventricular inflammation, perivascular fibrosis area, and microvessel density per field (magnification, ×200; lower panels). Scale bar = 100 μm. Error bars represent mean ± SEM (ventricular inflammation: No IR vs. WT IR + IgG p = 0.0003; WT IR + IgG vs. WT IR + Ab417 p = 0.0125; WT IR + IgG vs. p53KO IR + IgG p = 0.0104, perivascular fibrosis area: No IR vs. WT IR + IgG p = 0.0021; WT IR + IgG vs. WT IR + Ab417 p = 0.0328; WT IR + IgG vs. p 53KO IR + IgG p = 0.0203; WT IR + IgG vs. WT IR + Ab417 p = 0.0165, MVD: No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0055; WT IR + IgG vs. WT IR + Ab417 p = 0.0068). f Immunofluorescence staining (left panels) of α-SMA and CD31 in heart tissues 3 weeks post-irradiation ( n = 5 animals per group) and quantification (right panels) of the α-SMA + CD31 + area in the CD31 + area (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0251; WT IR + IgG vs. p53KO IR + IgG p = 0.011; p 53KO IR + IgG vs. p53KO IR + Ab417 p = 0.0004). g Immunofluorescence staining (left panel) of WGA, cTnT, and CD31 ( n = 5 animals per group) and quantification (right panel) of the cTnT area per field (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p = 0.0014; WT IR + IgG vs. WT IR + Ab417 p = 0.0489; WT IR + IgG vs. p53KO IR + IgG p = 0.0253; p53KO IR + IgG vs. p53KO IR + Ab417 p = 0.001). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant ( a Two-way ANOVA for multiple comparisons; all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Knockdown, Irradiation, Western Blot, Control, Immunofluorescence, Staining, Injection, Immunohistochemical staining

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a – e Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) three times a week for 2 weeks and received 16 Gy thoracic IR (No IR n = 7; IR + IgG n = 8; IR + Ab417 n = 8). f – j Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) with or without intraperitoneal Dox injection (4 mg/kg) three times a week for 2 weeks (No Dox n = 7; Dox+IgG n = 8; Dox+Ab417 n = 8). a , f Immunohistochemical detection (left panels) of L1-CT and γ-H2AX in heart tissues 1 week post IR ( a ) and 2 weeks after Dox treatment ( f ). The quantified L1-CT + cells and γ-H2AX + cells among ECs are shown (magnification, ×200; right panels). Scale bar = 50 μm. Error bars represent mean ± SEM (L1-CT: IR + IgG vs. IR + Ab417 p = 0.0004, γ-H2AX: No IR vs. IR + IgG p = 0.0076; IR + IgG vs. IR + Ab417 p = 0.0017; Dox + IgG vs. Dox+Ab417 p = 0.0003, **** p < 0.0001). b , g Haematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical detection of CD31 in heart tissues (left panels); and quantification of arterial wall thickness, perivascular fibrosis area, and microvessel density per field in heart tissues (magnification, ×200; right panels). Scale bar = 100 μm. The arrow in b indicates inflammatory cell infiltration. Error bars represent mean ± SEM (Arterial wall thickness: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0159; No Dox vs. Dox p = 0.0009; Dox + IgG vs. Dox + Ab417 p = 0.0117, perivascular fibrosis area: No IR vs. IR + IgG p = 0.0011; IR + IgG vs. IR + Ab417 p = 0.0168; No Dox vs. Dox p = 0.0001; Dox + IgG vs. Dox + Ab417 p = 0.0492, MVD: No IR vs. IR + IgG p = 0.0012; IR + IgG vs. IR + Ab417 p = 0.0184; No Dox vs. Dox p = 0.0093; Dox + IgG vs. Dox + Ab417 p = 0.0378). c , h Serum CRP, E-selectin, and ICAM-1 levels 1 week post IR ( c ) and 1 week after Dox treatment ( h ). Error bars represent mean ± SD (CRP: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0015; No Dox vs. Dox p = 0.0007; Dox+IgG vs. Dox+Ab417 p = 0.0003, E-selectin: No IR vs. IR + IgG p = 0.0031; IR + IgG vs. IR + Ab417 p = 0.02; No Dox vs. Dox p = 0.0003; Dox+IgG vs. Dox+Ab417 p = 0.0082, ICAM-1: No IR vs. IR + IgG p = 0.0053; IR + IgG vs. IR + Ab417 p = 0.01; No Dox vs. Dox p = 0.0154). d , i Immunofluorescence detection (upper panel) of L1-CT and CD31 in mouse heart tissues 1 week post IR ( d ) and 2 weeks after Dox treatment ( i ) (magnification, ×400). Scale bar = 5 μm. Quantification of L1CAM in CD31 nuclei (lower panel). Error bars represent mean ± SEM (No IR vs. IR + IgG p = 0.0012; IR + IgG vs. IR + Ab417 p = 0.0033; No Dox vs. Dox p < 0.0001; Dox IgG vs. Dox+Ab417 p = 0.0004). e , j Immunofluorescence staining of α-SMA and CD31 (scale bar = 10 μm) and of WGA and cTnT (scale bar = 20 μm) in heart tissues 1 week post IR ( e ) and 2 weeks post Dox treatment ( j ) (magnification, ×400; upper panels). Quantification of the α-SMA + CD31 + area in the CD31 + area and quantification of the cTnT area per field (lower panels). Error bars represent mean ± SEM (SMA + CD31 + area in the CD31 + area: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0011; No Dox vs. Dox p = 0.0013; Dox + IgG vs. Dox + Ab417 p = 0.0103, cTnT area: No IR vs. IR + IgG p = 0.0019; IR + IgG vs. IR + Ab417 p = 0.0107; No Dox vs. Dox p = 0.0126; Dox + IgG vs. Dox + Ab417 p = 0.0494, one-way ANOVA for multiple comparisons). Data are representative of three independent experiments.

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Injection, Control, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a , b Co-culture of iPSC-CMs and irradiated ECs transfected with control or L1CAM siRNA. a Schematic of the procedure for co-culture of iPSC-CMs and irradiated ECs. ECs were transfected with control or L1CAM-specific siRNA and irradiated with 10 Gy. Forty-eight hours after IR, the ECs were co-cultured with iPSC-CMs (top). The beating rates of the iPSC-CMs were examined after 5 days of co-culture with irradiated ECs (bottom). Error bars represent mean ± SD (No IR vs. si-Control+IR p = 0.0005; si-Control+IR vs. si-L1CAM + IR p > 0.0001). b Immunofluorescence staining (left panel) and quantification (right panel) of cTnT expression in cardiomyocytes and the collagen I deposition area in co-cultured cells (magnification, ×200). Scale bar = 50 μm. Error bars represent mean ± SD (cTnT expression: No IR vs. si-Control+IR p = 0.0239; si-Control+IR vs. si-L1CAM + IR p = 0.0475, collagen I deposition: No IR vs. si-Control + IR p = 0.027; si-Control + IR vs. si-L1CAM + IR p = 0492). c – e Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) three times a week for 2 weeks and received 16 Gy thoracic IR. f – h Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) with or without intraperitoneal Dox injection (4 mg/kg) three times a week for 2 weeks. c , f Cumulative survival analysis measured in days after treatment ( n = 8 animals per group). d , g Echocardiography results (upper panels) and quantification (lower panels) of FS (%), LVEF (%), LVESV (μL), and LVEDV (μL) (No IR n = 10; IR + IgG n = 5, IR + Ab417 n = 5; No Dox n = 10; Dox+IgG n = 5, Dox + Ab417 n = 5). Scale bar = 1 mm. Error bars represent mean ± SD (FS: No IR vs. IR + IgG p = 0.0331; IR + IgG vs. IR + Ab417 p = 0.0172; Dox+IgG vs. Dox + Ab417 p = 0.0046, LVEF: No IR vs. IR + IgG p = 0.0284; IR + IgG vs. IR + Ab417 p = 0.0182; Dox + IgG vs. Dox + Ab417 p = 0.0027, LVEDV: No IR vs. IR + IgG p = 0.0099; IR + IgG vs. IR + Ab417 p = 0.0054; No Dox vs. Dox p = 0.0003; Dox + IgG vs. Dox + Ab417 p = 0.003, LVESV: No IR vs. IR + IgG p = 0.0005; IR + IgG vs. IR + Ab417 p = 0.0002, **** p < 0.0001). e , h Haematoxylin and eosin–stained ventricular myocardium (upper panels) and quantification (lower panels) of cardiomyocyte cross-sectional area (No IR n = 7; IR + IgG n = 8; IR + Ab417 n = 8; No Dox n = 7; Dox + IgG n = 8; Dox+Ab417 n = 8) (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. IR + IgG p = 0.0003; IR + IgG vs. IR + Ab417 p = 0.0002; No Dox vs. Dos+IgG p = 0.024; Dox + IgG vs. Dox + Ab417 p = 0.0493). ( a , d : log-rank Mantel-Cox test; all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Co-Culture Assay, Irradiation, Transfection, Control, Cell Culture, Immunofluorescence, Staining, Expressing, Injection

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a Immunofluorescence staining of L1CAM and VE-cadherin in heart tissues from patients with cardiomyopathy ( n = 12) and patients without cardiomyopathy ( n = 8) (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM ( p = 0.0004). b Immunofluorescence staining of γ-H2AX and CD31 in heart tissues from patients with cardiomyopathy and patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM + cells among CD31 + cells (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM (Normal vs. γ-H2AX + CD31 + p > 0.0001; γ-H2AX ‒ CD31 + vs. γ-H2AX + CD31 + p = 0.0003) c Immunofluorescence staining of L1CAM, α-SMA, and CD31 in heart tissues from patients with cardiomyopathy and from patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM + cells among α-SMA - CD31 + and α -SMA + CD31 + cells (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM ( p > 0.0001). ( a two-tailed student’s t -test; b , c one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: LINC00115 links SETDB1 and PLK3 to regulate HIF1α expression. A Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled LINC00115. LINC00115 and anti- LINC00115 RNA were biotinylated by in vitro transcription, refolded, and incubated with MDA-MB-231 BCSCs total cell lysates. B WB of the proteins from antisense LINC00115 and LINC00115 pull-down assays. C Effects of LINC00115 KD on SETDB1 associated with PLK3 and HIF1α expression in MDA-MB-231 and B549 BCSCs. D Effects of LINC00115 KD on HIF1α expression with or without ectopic expression of HA-SETDB1 or/and Flag-PLK3 in MDA-MB-231 and B549 BCSCs. E A schematic model of LINC00115 functions in metastasis. LINC00115 may function as a scaffold lncRNA to recruit SETDB1 and PLK3 to activate the HIF1α signaling pathway. F Schematics of PLK3 full length (WT, 1-646aa), D1 (N-terminal, 1-315aa, contain Kinase domain (KD) mutant, and D2 [C-terminal, 316-646aa, contain polo box domain (PBD)] mutant plasmids. G Immunoblot analysis (IB) detection of SETDB1 interaction with PLK3 was immunoprecipitated with anti-Flag magnetic beads in HEK-293T cells transfected with the HA-SETDB1 and Flag-PLK3 constructs. H Schematics of SETDB1 full length (WT, 1-1291aa), M1 (N-terminal, 1-667aa, contain Tudor domain) mutant, and M2 (C-terminal, 672-1291aa, contain SET domain) mutant plasmids. I . IB of SETDB1 interaction with PLK3 and PLK3 methylation was immunoprecipitated with anti-HA magnetic beads in HEK-293T cells transfected with the Flag-PLK3 and HA-SETDB1 constructs

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: Expressing, Staining, Labeling, In Vitro, Incubation, Mutagenesis, Western Blot, Immunoprecipitation, Magnetic Beads, Transfection, Construct, Methylation

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: LINC00115 facilitates SETDB1 methylation of PLK3. A immunoprecipitation (IP) and IB analysis of PLK3 methylation in HEK293T cells treated with/without SETDB1 inhibitor TTD-IN (25 µM) for 12 h. B Mass spectrometry (MS) analysis showing potential methylation sites in PLK3 in BT549 and MDA-MB-231 BCSCs. A schematic representation of its amino acid sequence with all lysine (K) residues was highlighted in red. PB, Polo box domain. C LC-MS/MS spectrum of two tryptic peptides with a monomethylated residue at K106 and K200, carrying a mass of + 14.016 Da, respectively. D In vivo methylation assay of PLK3 using a pan anti-Lys-methyl antibody. Flag-tagged PLK3 WT or mutants were immunoprecipitated with anti-Flag magnetic beads from HEK293T cells co-expressed with HA-SETDB1. E Effects of methyltransferase activity-deficient mutants H1224K and C1226A of SETDB1 on PLK3 methylation in HEK293T cells. F Dot blot of PLK3-K106 or -K200 mono-methylation antibody using K106 or K200 unmodified (K106 or K200-free), and K106 or K200 monomethylated (K106 or K200-me1) peptides. G Effects of SETDB1 on PLK3 K106 and K200 methylation in MDA-MB-231/sgSETDB1 BCSCs transfected with SETDB1 sgRNA-resistant HA-SETDB1 WT , SETDB1 H1224K , or SETDB1 C1226A mutant

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: Methylation, Immunoprecipitation, Mass Spectrometry, Sequencing, Liquid Chromatography with Mass Spectroscopy, Residue, In Vivo, Magnetic Beads, Activity Assay, Dot Blot, Transfection, Mutagenesis

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: SETDB1 methylation of PLK3 is critical for BCSCs properties and metastasis. A Effects of ectopic expression of PLK3 WT or the 2KM mutant on mammospheres formation in MDA-MB-231/sh115 and BT549/sh115 BCSCs. B Limiting dilution mammosphere-forming assays of effects of ectopic expression of PLK3 WT or the 2KM mutant. C – F Effects of ectopic expression of PLK3 WT or the 2KM mutant on cell invasion ( C ), lung metastasis ( D , E ), and mouse lifespan ( F ). A cohort of athymic nude mice ( n = 6 per group) were implanted with 1 × 10 5 MDA-MB-231 BCSCs which were transfected with either control shRNA (shC) or sh115, with or without ectopic expression of PLK3 WT or PLK3 2KM mutant via the lateral tail vein. After 14 days, the mice were euthanized and the number of macroscopic lesions on the lung surfaces was quantified at necropsy. The cohorts consisted of mice injected with MDA-MB-231 /shC + EV, MDA-MB-231 /sh115 + EV, MDA-MB-231 /sh115 BCSCs with PLK3 WT or the 2KM mutant. Independent experimental groups under the same conditions were performed to calculate the animal survival ( n = 6). Scale bar, 100 μm. Data are representative of three independent experiments with similar results. Error bars, ± SEM. *** P < 0.001, by paired two-tail t -test or log-rank analysis

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: Methylation, Expressing, Mutagenesis, Transfection, Control, shRNA, Injection

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: SETDB1 methylation of PLK3 promotes PLK3 phosphorylation and decreases PLK3 activity. A IB of the phosphorylation and lysine methylation of PLK3, and also determined the expression of HIF1α, SOX2, and CD44 in control and SETDB1-knockdown MDA-MB-231 and BT549 BCSCs. Lysates were assessed by immunoprecipitation (IP) with anti-PLK3 and immunoblotting with anti-Phospho-(Ser/Thr) and anti-Lys-Mono-Methyl. B, C Effects of ectopic expression of PLK3 WT or 2KM (K106M/K200M) mutant on the phosphor-PLK3 and the expression of HIF1α, SOX2, and CD44 in MDA-MB-231 /sh115 ( B ) and MDA-MB-231/shSETDB1 BCSCs ( C ). EV, an empty vector control. Lysates were assessed by immunoprecipitation (IP) with anti-PLK3 and immunoblotting with anti-Phospho-(Ser/Thr). D In vitro GST pull-down analysis. Purified GST-HIF1α protein was incubated with cell extracts from MDA-MB-231 BCSCs. E and F Effects of ectopic expression of PLK3 WT or 2KM mutant on HIF-1α protein degradation in MDA-MB-231/shC or MDA-MB-231/sh115 BCSCs with cycloheximide (CHX, 100 µM) treatment (0, 10, 30, 60, and 120 min). G Effects of PLK3 WT or 2KM mutant on HIF-1α ubiquitination in MDA-MB-231/sh115 BCSCs. H Effects of ectopic expression of PLK3 WT or 2KM mutant on HIF-1α ubiquitination in MDA-MB-231/shSETDB1 BCSCs.Data are representative of two or three independent experiments with similar results. Error bars, ± SD. ** P < 0.01, by paired two-way Student’s t -test

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: Methylation, Phospho-proteomics, Activity Assay, Expressing, Control, Knockdown, Immunoprecipitation, Western Blot, Mutagenesis, Plasmid Preparation, In Vitro, Purification, Incubation, Ubiquitin Proteomics

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: HIFα, in turn, upregulates ALKBH5 to demethylate LINC00115 m 6 A and enhance LINC00115 stability. A WB of the proteins from antisense LINC00115 and LINC00115 pull-down assays. LINC00115 combined with ALKBH5 and YTHDF2. B A schematic model of LINC00115 stability regulated by ALKBH5 and YTHDF2. The stability of LINC00115 may be regulated through such a mechanism, which ALKBH5 demethylates of LIN00115 m 6 A and decreases YTHDF2-mediated m 6 A LIN00115 degradation. C Schematic representation of the position of m 6 A motifs within LINC00115 predicted by SRAMP. D m 6 A RNA immunoprecipitation (MeRIP)-qPCR was used to quantify relative LINC00115 m 6 A levels in ALKBH5 KD (shALKBH5-1 and shALKBH5-2) MDA-MB-231 and BT549 BCSCs. E QRT-PCR of LINC00115 levels in MDA-MB-231/shALKBH5and BT549/shALKBH5 BCSCs. F Interaction between YTHDF2 and LINC00115 was determined by RIP assay in MDA-MB-231 BCSCs with transfection of indicated DNAs. G QRT-PCR analysis of LINC00115 stability. MDA-MB-231 BCSCs and MDA-MB-231 YTHDF2 KD (shYTHDF2-1 and shYTHDF2-2) BCSCs were treated with Actinomycin D. H HIF1α knockdown impaired ALKBH5 expression in MDA-MB-231 and BT549 BCSCs. I Scatter plot of median standard-score-normalized (z-score) expression levels of HIF1α and ALKBH5 across human TNBC metastases after chemotherapy in the TCGA database ( n = 160). J Effects of ectopic expression of HIF1α on ALKBH5 expression in LINC00115 KD MDA-MB-231 and BT549 BCSCs. K Effects of ALKBH5 knockdown on SETDB1 association with PLK3. L Effects of ectopic expression of Flag-SETDB1 WT or the H1224K mutant on PLK3 methylation and HIF1α and ALKBH5 expression in MDA-MB-231 BCSCs/tetO-shSETDB1 and BT549 BCSCs/tetO-shSETDB1 cells. MDA-MB-231 and BT549 BCSCs with a tetO-shSETDB1 were pretreated with or without Dox. EV, an empty vector control. M, N Effects of ectopic expression of SETDB1 WT or H1224K mutant on LINC00115 m 6 A ( M ) and LINC00115 ( N ) levels. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. ** P < 0.01, by paired two-way Student’s t -test

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Transfection, Knockdown, Expressing, Mutagenesis, Methylation, Plasmid Preparation, Control

Journal: Molecular Cancer

Article Title: LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

doi: 10.1186/s12943-024-01975-3

Figure Lengend Snippet: Correlative expression of LINC00115, methylated-PLK3, and SETDB1 are prognostic. A Representative images of SETDB1, PLK3-K106me1, PLK3-K200me1, HIF1α, and LINC00115 in 118 clinical TNBC metastatic lymph node tissues. Scale bars, 50 μm. B Correlation of expression levels between SETDB1, PLK3K-106me1, PLK3K-200me1, HIF1α, and LINC00115. C Prognosis comparison of breast cancer patients with SETDB1/PLK3-K106me1 or -K200me1 ectopic differential expression using Kaplan-Meier survival analysis. D A working model of LINC00115 regulating chemoresistance breast cancer cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling. PTX therapy upregulates LINC00115 in BCSCs. LINC00115 functions as a scaffold lncRNA to link SETDB1 and PLK3 and enhance SETDB1 methylation of PLK3 at both K106 and K200. PLK3 methylation reduces phosphorylation of HIF1α and thereby increases HIF1α stability. HIF1α upregulates ALKBH5 to reduce m 6 A modification of LINC00115, resulting in decreased degradation of YTHDF2-dependent m 6 A-modified RNA and enhanced LINC00115 stability. Thus, this positive feedback loop provokes BCSC phenotypes, contributing to chemoresistance and metastasis. Targeting SETDB1 with a small molecular inhibitor reduces LINC00115-mediated BCSCs stemness and HIF1α expression and enhances BCSCs response to PTX.

Article Snippet: PLK3-K106 and -K200 mono-methylation rabbit polyclonal antibodies were raised by Proteintech (US) using the peptide E-K(me)-ILNEIELH and LGNFFITENMEL-K(me) (1:1000 for WB, 1: 50 for IHC).

Techniques: Expressing, Methylation, Comparison, Quantitative Proteomics, Phospho-proteomics, Modification

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Immunofluorescence of EU-RNA aggregates (gray) in iDUX4 cells that were left untreated (−DOX) or pulsed for 4-hours using 1ug/mL doxycycline (+DOX) and then incubated with 0.1mM 5-ethynyluridine (+EU) for 16-hours prior to washout and fixed at 24-hour time points (24–96 hours). Control cells were uninduced (−DOX) iDUX4 cells incubated with EU and fixed at either 24 hours or 96 hours or without EU and fixed at 24 hours. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( B ) Percent of cells with intranuclear EU-RNA aggregates in −DOX or +DOX iDUX4 cells labeled with EU for 16-hours at 24-hour time points. EU-RNA aggregates are present within +DOX iDUX4 cells from 24-hour to 72-hour time points. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± standard deviation (SD). Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU 24-hour time point). ( C ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. Housekeeping gene RPL27 is used as a control. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. ( D ) Quantitative RT-PCR of HSATII expression (2 −DCt ) in −DOX or +DOX iDUX4 cells treated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. EU-treatment does not impact HSATII RNA expression. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU). ( E ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours and harvested at 24-hour time points from 48-hours to 96-hours. Housekeeping gene RPL27 is used as a control. N=3. A total of three independent experimental replicates were performed. Data represent means ± SD. ( F ) Combined immunofluorescence and HSATII RNA-fluorescence in situ hybridization (RNA-FISH) of HSATII RNA (green) and EU-RNA (magenta) in +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( G ) Percent of cells with intranuclear EU-RNA aggregates only (“EU”), HSATII RNA aggregates only (“HSATII”) or both EU-RNA/ HSATII RNA aggregates (“both”) in +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( H ) Combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and EU-RNA (magenta) in control depleted (“CTRL KD”) or HSATII RNA-depleted (“HSATII KD”) +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( I ) Percent of cells with intranuclear RNA aggregates: EU only, HSATII only or both in CTRL KD or HSATII KD +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-way ANOVA Šídák’s multiple comparisons test between CTRL KD and HSATII KD within each group (EU, HSATII, both). Fig 1G and 1L : The frequency of cells containing RNA aggregates exhibits some variability across the population. This variability could be due to the robustness and induction efficiency of doxycycline treatment. ( J ) Combined immunofluorescence (IF) of EU-RNA (green) and Fibrillarin (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and fibrillarin (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( K ) Immunofluorescence (IF) of EU-RNA (green) and nucleophosmin-1 (NPM1) (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and NPM1 (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( L ) Percent of cells with nucleolar disruption present in cells with intranuclear RNA aggregates. N=2 experimental replicates. Minimum 300 nuclei per experiment.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Immunofluorescence, Incubation, Control, Labeling, Standard Deviation, Isolation, Quantitative RT-PCR, Expressing, RNA Expression, Fluorescence, In Situ Hybridization, Staining, Disruption

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Biological process pathway analysis of DUX4-induced stable EU-RNA associated RNPs (EU-RNPs) proteomics filtered protein hits (>2 unique peptide matches and >1.5 difference) ran through Enrichr. EU-RNP complexes were isolated from −DOX+EU, +DOX+EU or +DOX-EU iDUX4 cells harvested at the 48-hour time point using the “RICK” approach ( Bao et al., 2018 ). Protein was purified from isolated EU-RNP complexes and was subjected to mass-spectrometry. Proteomics was performed in experimental duplicates. ( B ) Validation of selected proteins identified through EU-RNP proteomics: Known DUX4-induced protein aggregates SC35 and TDP-43, and m 5 C-related factors NSUN2 and YBX-1. Proteins were isolated as stated in Fig. 2A . GAPDH and beta-tubulin were used as loading controls for inputs and negative controls for pulldown samples. 5% input was removed prior to RNA pulldown. N=1 per condition. A total of three independent experimental replicates were performed. −D+E, −DOX+EU samples; +D+E, +DOX+EU samples; +D-E, +DOX-EU samples. Quantification of immunoblot is shown right. ( C ) Immunofluorescence of EU-RNA (green) and SC35 (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( D ) Mean signal intensity of SC35 within specified regions of interest (ROI) in the nucleus: either within EU-RNA foci or the remainder of the nucleoplasm in +DOX EU-RNA+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. N= 50 ROI.. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( E ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and SC35 (magenta) in −DOX or +DOX iDUX4 cells fixed at 24-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( F ) Mean signal intensity of SC35 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 35 ROI.. A total of two independent experimental replicates were performed. Data represent means. ( G ) Immunofluorescence of EU-RNA (green) and TDP-43 (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( H ) Mean signal intensity of TDP-43 within specified ROI in the nucleus: either within EU-RNA foci or the remainder of the nucleoplasm in +DOX EU-RNA+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. N= 30 ROI.. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( I ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and TDP-43 (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( J ) Mean signal intensity of TDP-43 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 90 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( K ) Percent of cells with TDP-43 nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Isolation, Purification, Mass Spectrometry, Biomarker Discovery, Western Blot, Immunofluorescence, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Experimental schematic showing ChIRP protocol ( Chu et al., 2012 ). ( B ) RT-qPCR showing enrichment of RPL27 , ZSCAN4 or HSATII RNA in ChIRP pulldowns using either control ASOs or HSATII-specific ASOs in +DOX iDUX4 cells. N=3 biological replicates per experiment (small black points). A total of three independent experimental replicates were performed (colored points). ( C ) Immunoblot showing enrichment of known HSATII interacting proteins MeCP2 and eIF4A3 in ChIRP pulldowns (PD) using either control ASOs (CTRL) or HSATII-specific ASOs (HSATII) in −DOX or +DOX iDUX4 cells. GAPDH used as loading control for inputs and negative control for PD samples. 5% input was removed prior to RNA pulldown. Immunoblot shows two biological replicates. N=2 per condition. A total of three experimental replicates were performed. Quantification of immunoblot is shown below. ( D ) Biological process pathway analysis of HSATII RNA associated RNPs (HSATII-RNP) proteomics filtered protein hits (>2 unique peptide matches and >1.5 difference) ran through Enrichr. HSATII-RNP complexes were isolated from −DOX or +DOX iDUX4 cells harvested at the 24-hour time point using the “ChIRP” approach ( Chu et al., 2012 ). Protein was purified from isolated HSATII-RNP complexes using antisense oligonucleotides (ASO) targeting HSATII or control sequences and was subjected to mass-spectrometry. Proteomics was performed in biological triplicate. ( E ) Validation of selected proteins identified through HSATII-RNP proteomics: m 5 C-related factors YBX-1 and m 6 A-related factors YTHDC1, WTAP, and VIRMA. Proteins were isolated as stated in Fig. 3B . GAPDH was used as a loading control for inputs and negative control for pulldown samples. 5% input was removed prior to RNA pulldown. N=1 per condition. A total of three independent experimental replicates were performed.. CTRL, Control ASO; HSATII, HSATII-specific ASO; PD, pulldown. Quantification of immunoblot is shown right.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Quantitative RT-PCR, Control, Western Blot, Negative Control, Isolation, Purification, Mass Spectrometry, Biomarker Discovery

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and VIRMA (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( B ) Percent of nuclei with VIRMA signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( C ) Mean signal intensity of VIRMA within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 60 ROI. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( D ) Percent of cells with VIRMA nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed t-test. ( E ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and WTAP (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( F ) Percent of nuclei with WTAP signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( G ) Mean signal intensity of WTAP within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 60 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( H ) Percent of cells with WTAP nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed Unpaired t-test. ( I ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YTHDC1 (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( J ) Percent of nuclei with YTHDC1 signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. Dots represent fields taken from representative experiment. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( K ) Mean signal intensity of YTHDC1 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 25 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Unpaired t-test. ( L ) Percent of cells with YTHDC1 nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed Unpaired t-test. ( M ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and m 6 A (magenta) in −DOX or +DOX iDUX4 cells fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( N ) Mean signal intensity of m 6 A within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 100 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann-Whitney test. ( O ) Mean signal intensity of nuclear m 6 A signal in −DOX or +DOX cells fixed at 24-hour time point. Dots are individual nuclei. N= 100 nuclei. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing One-way ANOVA Tukey’s multiple comparison test. ( P ) RNA dot blot of RNA isolated from uninduced (−DOX), control depleted (siCTRL), VIRMA depleted (siVIRMA) or +DOX iDUX4 cells harvested at 24-hours. 200ng of RNA was loaded. RNA was probed using anti-m 6 A antibody or total RNA was determined using Methylene Blue. Signal intensity showed an increase in m 6 A RNA levels in +DOX iDUX4 cells compared to uninduced cells. N=3.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Immunofluorescence, MANN-WHITNEY, Two Tailed Test, Comparison, Dot Blot, Isolation, Control

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and NSUN2 (magenta) in −DOX or +DOX iDUX4 cells fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( B ) Fraction of nuclei with NSUN2 signal that at least have partial signal overlap with HSATII RNA foci in +DOX iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( C ) Mean signal intensity of NSUN2 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 50 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Unpaired t-test. ( D ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and m 5 C (magenta) in +DOX iDUX4 cells either pre-treated with DMSO or 5μM 5-azaC for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( E ) Mean signal intensity of nuclear m 5 C signal in −DOX or +DOX HSATII negative (HSATII−) or HSATII positive (HSATII+) cells fixed at 24-hour time point. Dots are individual nuclei. N= 50 nuclei. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing One-way ANOVA Tukey’s multiple comparison test. ( F ) Mean signal intensity of m 5 C within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in untreated or 5-azaC treated +DOX iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 50 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing one-way ANOVA Tukey’s multiple comparison test. ( G ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD and fixed 24-hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( H ) Percent of nuclei with YBX-1 signal that at least have partial signal overlap with HSATII RNA foci in +DOX iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( I ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells fixed 24-hours post-induction. Scale bar = 20μm. Image is representative of three independent experimental replicates performed. ( J ) Cytoplasmic signal intensity of YBX-1 in −DOX or +DOX iDUX4 cells that have no YBX-1 nuclear aggregates (YBX-1−) or with YBX-1 nuclear aggregates (YBX-1+). N= 200 nuclei. A total of three independent experimental replicates were performed. Data represent means ± SD. Statistical differences between −DOX (control) and +DOX iDUX4 cells were analyzed employing one-way ANOVA Tukey’s multiple comparison test. ( K ) Nuclear mean signal intensity of YBX-1 within −DOX, +DOX HSATII−, +DOX HSATII+ or +DOX HSATII KD iDUX4 nuclei. Dots indicate individual nuclei. N= 40 nuclei. A total of three independent experimental replicates were performed. Statistical differences between −DOX (control) and +DOX iDUX4 cells were analyzed employing one-way ANOVA Tukey’s multiple comparison test. ( L ) Cytoplasmic mean signal intensity of YBX-1 within −DOX CTRL, −DOX HSATII KD, +DOX CTRL or HSATII KD iDUX4 cells. N= 50 cells. A total of three independent experimental replicates were performed. Statistical differences between samples were analyzed employing one-way ANOVA Tukey’s multiple comparison test. ( M ) Mean signal intensity of YBX-1 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 50 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( N ) Percent of cells with YBX-1 nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Unpaired t-test. ( O ) Immunoblot of whole cell lysate from −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD and harvested at 24-hours post-induction. Blot was probed for YBX-1 total protein levels. GAPDH was used as loading control. Quantification of YBX-1 relative protein levels are indicated above blot. N=1 per condition.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Immunofluorescence, Comparison, Control, MANN-WHITNEY, Western Blot

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells either pre-treated with DMSO or 5μM 5-azaC for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 10μm. Images are representative of three independent experimental replicates performed. ( B ) Percent of HSATII+ nuclei with YBX-1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells with or without 5-azaC treatment. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test. ( C ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and m 5 C (magenta) in +DOX iDUX4 cells either pre-treated with siRNAs targeting control sequences (siCTRL) or NSUN2 (siNSUN2) for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( D ) Mean signal intensity of m 5 C within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 50 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing one-way ANOVA Tukey’s multiple comparison test. ( E ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells either pre-treated with siRNAs targeting control sequences (siCTRL) or NSUN2 (siNSUN2) for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( F ) Percent of HSATII+ nuclei with YBX-1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing Mann Whitney test. ( G ) Percent of nuclei with YBX-1 nuclear aggregates in +DOX iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Mann Whitney test.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Activity Assay, Immunofluorescence, Two Tailed Test, Control, Comparison, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Top panel: combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells and fixed at either 16-hour time point or 24-hour time point; Bottom panel: Immunofluorescence of dsRNA (green) and YBX-1 (magenta) in +DOX iDUX4 cells and fixed at either 16-hour time point or 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( B ) Frequency of nuclei with either HSATII ssRNA or dsRNA in −DOX or +DOX iDUX4 cells at either 16-hour time point or 24-hour time point. N=2–5 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( C ) Percent of HSATII+ nuclei with YBX-1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells at either 16-hour time point or 24-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing Two-tailed Unpaired t-test. ( D ) Percent of dsRNA+ nuclei with YBX-1 signal that overlap with dsRNA foci in +DOX iDUX4 cells at either 16-hour time point or 24-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( E ) Mean signal intensity of YBX-1 within specified ROI in the nucleus: either within dsRNA foci or the remainder of the nucleoplasm in +DOX dsRNA+ iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as dsRNA foci ROI. N= 30 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test. ( F ) Top panel: combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YTHDC1 (magenta) in +DOX iDUX4 cells and fixed at either 16-hour time point or 24-hour time point; Bottom panel: Immunofluorescence of dsRNA (green) and YTHDC1 (magenta) in +DOX iDUX4 cells and fixed at either 16-hour time point or 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( G ) Percent of HSATII+ nuclei with YTHDC1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells at either 16-hour time point or 24-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( H ) Percent of dsRNA+ nuclei with YTHDC1 signal that overlap with dsRNA foci in +DOX iDUX4 cells at either 16-hour time point or 24-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( I ) Percent of nuclei with HSATII reverse RNA signal (HSATII rv+), HSATII forward RNA signal (HSATII fwd+) or dsRNA signal (K1+) in +DOX iDUX4 cells treated with control gapmers (CTRL KD) or HSATII forward-specific gapmers (HSATII forward-strand KD). N=3 experimental replicates. Minimum 450 nuclei per experiment. Data represent means ± SD. ( J ) Mean signal intensity of nuclear YBX-1 within uninduced control depleted iDUX4 cells, or within HSATII rv+ nuclei in either induced control depleted or HSATII forward-strand depleted iDUX4 cells. N= 50 nuclei. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing One-way ANOVA Tukey’s multiple comparison test.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Immunofluorescence, Two Tailed Test, Control, Comparison

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) RNA processing consequences and fraction of genes with indicated consequence in control depleted (CTRL) −DOX or +DOX iDUX4 cells or in +DOX iDUX4 cells with control (CTRL KD) or HSATII depletion (HSATII KD). RNA-sequencing was performed in biological duplicate. UTR: untranslated region; SE: skipped exon; IR: intron retention; MES: mutually exclusive splicing; ATSS: alternative transcription start site; MEE: mutually exclusive exon; ATTS: alternative transcription termination site; FDR: false discovery rate. ( B ) Top enrichment of Biological Processes of significantly affected genes between +DOX iDUX4 CTRL KD compared to HSATII KD. Ratio indicated number of genes within pathway. ( C ) UCSC genome browser tracks showing RNA-seq reads which map to gene Trip12 from −DOX iDUX4 cells with Ctrl (blue) or HSATII-depletion (green) or +DOX iDUX4 cells with Ctrl (red) or HSATII-depletion (purple). +DOX Ctrl cells have increased intron retention (highlighted region, orange bar). HSATII depletion decreases intron retention. ( D ) Expression of Trip12 with intron retention in −DOX or +DOX iDUX4 cells with (HSATII KD) or without (CTRL KD) HSATII depletion. N= 3. Data represent means ± SD. Statistical differences between groups were analyzed employing 2-way ANOVA Tukey’s multiple comparison test. ( E ) UCSC genome browser tracks showing RNA-seq reads which map to gene Slc25a44 from −DOX iDUX4 cells with Ctrl (blue) or HSATII-depletion (green) or +DOX iDUX4 cells with Ctrl (red) or HSATII-depletion (purple). +DOX Ctrl cells have decreased exon 2 and intron inclusion (highlighted region, orange bar). HSATII depletion increases exon 2 retention and intron inclusion. ( F ) Expression of Slc25a44 containing exon 2 (left panel) or intron retaining (right panel) isoforms −DOX or +DOX iDUX4 cells with (HSATII KD) or without (CTRL KD) HSATII depletion. N= 3. Data represent means ± SD. Statistical differences between groups were analyzed employing 2-way ANOVA Tukey’s multiple comparison test.

Article Snippet: Immortalized MB135 myoblast cells ( H. sapiens , female, Fields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center, https://www.urmc.rochester.edu/neurology/fshd-center.aspx ) that contain a tet-inducible DUX4 transgene (iDUX4) (RRID:Addgene_99281) ( Resnick et al., 2019 ) were cultured in F10 medium (Gibco/ ThermoFisher Scientific) supplemented with 20% Fetal Bovine Serum (GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Gibco/ ThermoFisher Scientific), 10ng/mL recombinant human FGF (PeproTech) and 1μM dexamethasone (Sigma-Aldrich). iDUX4 cells were induced in the presence of 2μg/mL of doxycycline-hyclate (“DOX”; Sigma-Aldrich) for 4-hrs and assayed 20-hrs post-induction, unless otherwise noted.

Techniques: Expressing, Control, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative Proteomics, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative western blot and relative quantification of the high-glycosylated (50kDa) (B) and the low-glycosylated (37kDa) (C) forms of EMMPRIN (EMN) in the ventral horns of the lumbar spinal cord of Ntg (black dots) and SOD1 G93A (red dots) mice. Two-Way Anova: HG-EMN (interaction, p=0.084; age, p<0.0001; genotype, p<0.0001) followed by T-test for genotype comparison: HG-EMN (PS, p=0.4986; ON, **, p=0.0064; SY, ***, p=0.0005; ES, **, p=0.0052); LG-EMN (interaction, p=0.6868; age, p=0.825; genotype, p=0.831). One-Way Anova for linear trend: HG-EMN, p<0.0001; LG-EMN, p=0.0636. Target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=5 mice/stage. (D) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at the onset of the disease. Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. Experiments have been performed in n=3 mice/group. (E) Representative image of EMMPRIN (EMN, gray) expression in astrocytes (GFAP, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. (F) Representative image of EMMPRIN (EMN, gray) expression in microglia (Iba1, red) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. Of note, E and F are the same image but with split channels and performed appropriate merge. (G) Relative quantification of the percentage of astrocytes (GFAP) or microglia (Iba1) expressing EMMPRIN. Two-Way Anova (interaction, p=0.0003; age, p<0.0001; cell type, p<0.0001) followed by Bonferroni multiple comparison test for cell type comparison (***PS, p=0.0001; ON, SY, ES ****, p<0.0001). One-Way Anova for linear trend: GFAP, p<0.0001; Iba1, p<0.0001. Data are mean±SEM of n=4-5 mice/stage. For all experiment: PS, presymptomatic; ON, onset; SY, symptomatic; ES, end-stage. For D-F scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Comparison, Expressing, Labeling

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative image of primary cultures of NTg and SOD1 G93A astrocytes. Most of the cells present in the preparation are astrocytes (GFAP), and only a very small percentage of microglia (Iba1) cells is detected. Scale bar = 100μm. (B) Representative western blot and (C) relative quantification of EMMPRIN (EMN) in NTg astrocytes treated 24h with 0.5nM recombinant PPIA. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05 by unpaired T-Test. (D) Relative quantification of factors released by NTg astrocytes after 24h of treatment with 0.5nM recombinant PPIA (N:P) compared to untreated NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of NTg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 1. (E) Pie chart of upregulated, unchanged or downregulated proteins in NTg astrocytes treated with PPIA compared to untreated NTg astrocytes. (F) Significant leading pathways related to the 43 upregulated proteins found in NTg astrocytes treated with PPIA. Proteins associated with the pathways are listed in Supplementary Table 2.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Recombinant

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Representative dot blot (A) and relative quantification ( B) of extracellular PPIA in the CSF of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. *, p=0.0248. One-Way Anova for linear trend in TDP-43 A315T mice: p=0.1466. Representative western blot ( C ) and relative quantification of the high-glycosylated (50kDa) (D) and the low-glycosylated (37kDa) (E) forms of EMMPRIN (EMN) in the lumbar spinal cord of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. **, p=0.0020; ***, p=0.0001. One-Way Anova for linear trend in TDP-43 A315T mice: HG-EMN, p=0.0051; LG-EMN, p=0.0054. For A-E, target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=3-4 mice/stage (6 months, onset; 10 months, early symptomatic; 13 months, late-symptomatic). (F) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and TDP-43 A315T mice at the onset of the disease (6 months). Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. (G) Representative image of EMMPRIN (EMN, green) expression in astrocytes (GFAP, red) or microglia (Iba1, gray) in the ventral horn of the lumbar spinal cord of TDP-43 A315T mice at the early symptomatic stage (10 months). Please note that due to antigen retrieval we observed some Iba1 leaking signal in neurons (yellow arrow heads). For F, G: experiments have been performed in n=3 mice/group. Scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Dot Blot, Quantitative Proteomics, Comparison, Western Blot, Expressing, Labeling

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative Proteomics, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative western blot and relative quantification of the high-glycosylated (50kDa) (B) and the low-glycosylated (37kDa) (C) forms of EMMPRIN (EMN) in the ventral horns of the lumbar spinal cord of Ntg (black dots) and SOD1 G93A (red dots) mice. Two-Way Anova: HG-EMN (interaction, p=0.084; age, p<0.0001; genotype, p<0.0001) followed by T-test for genotype comparison: HG-EMN (PS, p=0.4986; ON, **, p=0.0064; SY, ***, p=0.0005; ES, **, p=0.0052); LG-EMN (interaction, p=0.6868; age, p=0.825; genotype, p=0.831). One-Way Anova for linear trend: HG-EMN, p<0.0001; LG-EMN, p=0.0636. Target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=5 mice/stage. (D) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at the onset of the disease. Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. Experiments have been performed in n=3 mice/group. (E) Representative image of EMMPRIN (EMN, gray) expression in astrocytes (GFAP, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. (F) Representative image of EMMPRIN (EMN, gray) expression in microglia (Iba1, red) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. Of note, E and F are the same image but with split channels and performed appropriate merge. (G) Relative quantification of the percentage of astrocytes (GFAP) or microglia (Iba1) expressing EMMPRIN. Two-Way Anova (interaction, p=0.0003; age, p<0.0001; cell type, p<0.0001) followed by Bonferroni multiple comparison test for cell type comparison (***PS, p=0.0001; ON, SY, ES ****, p<0.0001). One-Way Anova for linear trend: GFAP, p<0.0001; Iba1, p<0.0001. Data are mean±SEM of n=4-5 mice/stage. For all experiment: PS, presymptomatic; ON, onset; SY, symptomatic; ES, end-stage. For D-F scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Comparison, Expressing, Labeling

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative image of primary cultures of NTg and SOD1 G93A astrocytes. Most of the cells present in the preparation are astrocytes (GFAP), and only a very small percentage of microglia (Iba1) cells is detected. Scale bar = 100μm. (B) Representative western blot and (C) relative quantification of EMMPRIN (EMN) in NTg astrocytes treated 24h with 0.5nM recombinant PPIA. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05 by unpaired T-Test. (D) Relative quantification of factors released by NTg astrocytes after 24h of treatment with 0.5nM recombinant PPIA (N:P) compared to untreated NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of NTg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 1. (E) Pie chart of upregulated, unchanged or downregulated proteins in NTg astrocytes treated with PPIA compared to untreated NTg astrocytes. (F) Significant leading pathways related to the 43 upregulated proteins found in NTg astrocytes treated with PPIA. Proteins associated with the pathways are listed in Supplementary Table 2.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Recombinant

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Representative dot blot (A) and relative quantification ( B) of extracellular PPIA in the CSF of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. *, p=0.0248. One-Way Anova for linear trend in TDP-43 A315T mice: p=0.1466. Representative western blot ( C ) and relative quantification of the high-glycosylated (50kDa) (D) and the low-glycosylated (37kDa) (E) forms of EMMPRIN (EMN) in the lumbar spinal cord of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. **, p=0.0020; ***, p=0.0001. One-Way Anova for linear trend in TDP-43 A315T mice: HG-EMN, p=0.0051; LG-EMN, p=0.0054. For A-E, target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=3-4 mice/stage (6 months, onset; 10 months, early symptomatic; 13 months, late-symptomatic). (F) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and TDP-43 A315T mice at the onset of the disease (6 months). Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. (G) Representative image of EMMPRIN (EMN, green) expression in astrocytes (GFAP, red) or microglia (Iba1, gray) in the ventral horn of the lumbar spinal cord of TDP-43 A315T mice at the early symptomatic stage (10 months). Please note that due to antigen retrieval we observed some Iba1 leaking signal in neurons (yellow arrow heads). For F, G: experiments have been performed in n=3 mice/group. Scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Dot Blot, Quantitative Proteomics, Comparison, Western Blot, Expressing, Labeling