Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Differential gene expression analysis of synovial myeloid (CD11b + Gr1 + ) populations from immunized wild-type (wt) and Fas-deficient ( Fas −/− ) mice on day 10 of antigen-induced arthritis. Volcano plots showing negative logarithm of adjusted p values (-log 10 p), in relation to the logarithm of gene expression fold-change (log 2 FC) for differential gene expression analysis between mice with arthritis (AIA) and immunized control mice (IMM) (A) and Fas −/− mice and wt mice (B) . Markers of individual genes with significant adjusted p values (Benjamini-Hochberg, BH, p < 0.05) and fold change (absolute FC > 1.5) are marked red. (C) Validation of microarray data by RT-PCR analysis of Erdr1 , Mid1 , and Thbs1 expression in pooled sorted CD11b + Gr-1 + cells from wt (n = 5), and Fas −/− mice (n = 4) with arthritis (AIA), and knee tissue extracts (D) of wt (n = 6) and Fas −/− AIA mice (n = 7, right panels). Samples for microarray were selected from three separate experiments, based on the RNA quality. Validation of microarray results was performed in two separate experiments, one for analysis of sorted cells and one for the analysis of tissue extracts. Mice were sacrificed on day 10 after arthritis induction. Gene expression is normalized to the β-actin expression. Horizontal line, median; boxes, IQR; whiskers, range; statistical significance is stated on plots (p < 0.05, Mann-Whitney test).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Gene Expression, Control, Biomarker Discovery, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Expression of Mid-1 over the course of AIA. (A) Expression of Mid1 RNA in the knee joints of wt and Fas −/− mice with arthritis, assessed by in situ hybridization (RNAScope). Arrows, brown signal resulting from binding of Mid1 probe, bars, 50 µm. Slides for RNA Scope were selected from a representative experiment used for histology and µCT analysis. Three joints from wt and Fas −/− mice with arthritis were hybridized, with similar results, and representative images are shown. (B) Western blot analysis of Mid-1 expression in knee tissue extracts of wt and Fas −/− AIA mice, and analysis of Mid-1 signal intensity, normalized to total protein signal intensity, acquired from membranes using stain-free technology . Protein extraction was performed from 6 wt and 2 Fas−/− knees harvested from one experiment. (C) Expression of Mid1 in various tissues of wt (n = 2) and Fas −/− mice (n = 2) with AIA. Tissues were harvested from a single experiment. (D) Expression of Mid1 and IL-1β in total joint tissue extracts of wt mice during immunization (d3-21), early (d24), and 1 week after arthritis induction (d28) in a single experiment. Gene expression was determined by RT-PCR and normalized to the expression of β-actin , n = 3, markers, individual values, horizontal lines, median (IQR), * denotes p < 0.05, in comparison to NI group (Kruskal-Wallis test).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Expressing, In Situ Hybridization, RNAscope, Binding Assay, Western Blot, Staining, Protein Extraction, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Myeloid-specific ablation of Fas does not ameliorate antigen-induced arthritis (AIA). Fas fl/fl LysMCre +/− were crossed with Fas fl/fl mice to produce littermates with conditional ablation of Fas in myeloid cells (Fas fl/fl LysMCre +/− , Cre + ) and controls (Fas fl/fl LysMCre −/− , Cre – ). Experiments were repeated 3 times and data are cumulative of minimum 2 representative experiments. (A) Bone marrow cells from Cre − , Cre + and Fas −/− mice were stained with anti-Fas(CD95)-AF488 or corresponding isotype control, and CD11b-PE, Gr-1-PECy7, F4/80-APCCy7, and CD3/B220/NK1.1-APC antibodies. Mean fluorescence intensities (MFI) for each population are shown as individual values (markers), horizontal lines and bars are mean ± SD. Mice were sacrificed on a day 10 post-i.a. injection and arthritis was assessed by (B) measuring knee diameters and semi quantitative visual soring using appropriate scale (0-no arthritis, 1-discrete localized thickening of the joint capsule, 2-mild swelling, absence of sharp patellar ligament contour, 3-clear swelling with diffuse thickening of the joint capsule, 4-severe swelling and deformity, visible through the skin). (C) Subchondral epiphyseal bone volume was assessed by µCT in Cre-NI − (n = 7 4m/3f), Cre − AIA (n = 13, 5m/8f), Cre + NI (n = 6, 4m/2f), Cre + AIA (n = 14, 10m/4f). The following variables were analyzed in distal femoral epiphyses: trabecular bone volume (BV/TV, %), trabecular number (Tb.N., mm -1 ), trabecular thickness (Tb.Th., µm), and trabecular separation (Tb.Sep., µm). (D) Expression of Fas, TNF, IL-1β and Mid1 mRNA in total knee joint tissue from Cre − mice with AIA (Cre − , n = 4, 2m/2f), and Cre + mice with AIA (Cre + , n = 6, 4m/2f). Markers represent individual values, horizontal lines and error bars are mean ± SD (A, B top panel, C) or median (IQR) (B bottom panel, D); statistical significance is marked on plots with red lines connecting experimental groups with p < 0.05 (ANOVA and Student-Newman-Keuls post hoc test, A, B top panel, C; Kruskal-Wallis test, B bottom panel; Mann-Whitney test, D).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Staining, Control, Fluorescence, Injection, Expressing, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Proinflammatory and apoptotic effect of anti-Fas antibody on cells of myeloid (CD11b + Gr1 + ) lineage. Inflammation was induced by 2 μg/mL heat-inactivated Mycobacterium tuberculosis , and after incubation for 1 h/37°C, appropriate combinations of anti-Fas antibody, isotype control antibody, and protein G were added. After incubation for 18h/37°C cells were harvested for flow cytometry and/or RNA isolation. (A) Proportion of apoptotic (annexin V + ) cells, and expression of proinflammatory IL-1β and Mid1 in groups of non-stimulated (NS) and inflammatory-stimulated cells (S) treated with 1 μg/ml isotype control antibody (NI IgG) and/or 1 μg/ml protein G (Prot G) and/or 1 μg/ml anti-Fas antibody (aFas), (B) Expression of proinflammatory cytokines in groups of non-stimulated (NS) and inflammatory-stimulated cells (S) treated with low doses (0.01–0.1 μg/ml) of anti-Fas antibody. 10 6 cells were plated per well of a 96-well culture plate. Cells were grown in duplicates (n = 2, A) or triplicates (n = 3, B), and experiments were repeated three times with similar results. Markers, individual values, horizontal lines, mean ± SD. Statistically significant difference within groups of S and NS cells is marked with red lines connecting experimental groups with p < 0.05, statistically significant difference between identically treated S and NS cells is marked with blue lines connecting experimental groups with p < 0.05, p values are marked on plots (ANOVA and Student-Newman-Keuls post-hoc test).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Incubation, Control, Flow Cytometry, Isolation, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Effects of Mid-1 inactivation on the expression of pro-inflammatory cytokines in inflammatory-stimulated cells. (A) Pharmacological inhibition of Mid-1 in inflammatory-stimulated bone marrow cells. Cells were pre-treated with 20 µM metformin or 20 µM of Mid-1 peptide antagonist (GSK'364A) for 1h/37°C which was followed by the addition of heat inactivated Mycobacterium tuberculosis in final concentration of 10 µg/µL. After overnight incubation at 37°C, expression of proinflammatory cytokines IL-1β , and TNF and concentration of TNF -α were assessed by RT-PCR and ELISA. Experiments were performed in duplicates to quadruplicates, and values are calculated as ratios of means of expression/concentration of cytokine in the stimulated cells treated with metformin or GSK'364A over means of expression/concentration of cytokine in stimulated untreated cells in each experiment (n = 3). (B) Expression of inflammatory cytokines in inflammatory-stimulated bone marrow cells from wt and Mid1 −/− mice treated with agonistic anti-Fas antibody. Cells were treated with 10 µg/µL heat-inactivated Mycobacterium tuberculosis and 0.1 µg/mL anti-Fas antibody. After overnight incubation at 37°C, expression of proinflammatory cytokines IL-1β , and TNF and concentration of TNF -α were assessed by RT-PCR and ELISA. Experiments were performed in duplicates to quadruplicates, and values are calculated as ratios of means of expression/concentration of cytokine in the stimulated cells treated with metformin or GSK'364A over means of expression/concentration of cytokine in stimulated untreated cells in each experiment (n = 3). Horizontal line, median; boxes, IQR; whiskers, range; p values are marked on plots (A, Kruskal-Wallis test; B, Mann- Whitney test).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Expressing, Inhibition, Concentration Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Mid-1 inactivation moderately alleviates antigen-induced arthritis (AIA). (A) AIA was induced in mice with a gene knockout for Mid1 ( Mid1 −/− ) and wt mice. Visual score, knee diameters and µCT values represent cumulative data from 4 separate experiments, while histology score was assessed in one of the representative experiments. Mice were sacrificed on a day 10 post-i.a. injection and arthritis was assessed by (B) measuring knee diameters and semi quantitative visual soring in wt NI (n = 21), wt AIA (n = 23), Mid1 −/− NI (n = 10), Mid1 −/− AIA (n = 20) using appropriate scale (0-no arthritis, 1-discrete localized thickening of the joint capsule, 2-mild swelling, absence of sharp patellar ligament contour, 3-clear swelling with diffuse thickening of the joint capsule, 4-severe swelling and deformity, visible through the skin). (C) Total histology score in wt NI (n = 6), wt AIA (n = 6), Mid1 −/− NI (n = 4), and Mid1 −/− AIA (n = 9). Scoring was performed according to synovial thickening, presence of exudate in joint space, cartilage degradation, and subchondral bone damage, according to the 3 point scale. (D) Subchondral epiphyseal bone volume was assessed by µCT in wt NI (n = 21), wt AIA (n = 23), Mid1 −/− NI (n = 10), Mid1 −/− AIA (n = 20). The following variables were analyzed in distal femoral epiphyses: trabecular bone volume (BV/TV, %), trabecular number (Tb.N., mm -1 ), trabecular thickness (Tb.Th., µm), and trabecular separation (Tb.Sep., µm). (E) Representative 3D models of distal femora, from µCT reconstruction images. Markers represent individual values, horizontal lines and error bars are mean ± SD (B left panel, C) or median (IQR) (B right panel, D); statistical significance is marked on plots with red lines connecting experimental groups with p < 0.05 (ANOVA and Student-Newman-Keuls post hoc test, B left panel, C; Kruskal-Wallis test, B right panel, D).

Article Snippet: Total RNA was converted to cDNA by reverse transcriptase (Thermo Fisher Scientific), and amplified by an ABI 7500 instrument (Applied Biosystems, Thermo Fisher Scientific), using commercially available TaqMan Assays (mouse β-actin , Mm00607939_s1; Erdr1 Mm04214945_uH; Fas , Mm00487425_m1; IL-1β , Mm00434228_m1; IL-6 , Mm00446190_m1; Mid 1, Mm01166432_m1 Thbs1 Mm00449018_m1; TNF , Mm00443258_m1), as described previously ( ).

Techniques: Gene Knockout, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Differential gene expression analysis of synovial myeloid (CD11b + Gr1 + ) populations from immunized wild-type (wt) and Fas-deficient ( Fas −/− ) mice on day 10 of antigen-induced arthritis. Volcano plots showing negative logarithm of adjusted p values (-log 10 p), in relation to the logarithm of gene expression fold-change (log 2 FC) for differential gene expression analysis between mice with arthritis (AIA) and immunized control mice (IMM) (A) and Fas −/− mice and wt mice (B) . Markers of individual genes with significant adjusted p values (Benjamini-Hochberg, BH, p < 0.05) and fold change (absolute FC > 1.5) are marked red. (C) Validation of microarray data by RT-PCR analysis of Erdr1 , Mid1 , and Thbs1 expression in pooled sorted CD11b + Gr-1 + cells from wt (n = 5), and Fas −/− mice (n = 4) with arthritis (AIA), and knee tissue extracts (D) of wt (n = 6) and Fas −/− AIA mice (n = 7, right panels). Samples for microarray were selected from three separate experiments, based on the RNA quality. Validation of microarray results was performed in two separate experiments, one for analysis of sorted cells and one for the analysis of tissue extracts. Mice were sacrificed on day 10 after arthritis induction. Gene expression is normalized to the β-actin expression. Horizontal line, median; boxes, IQR; whiskers, range; statistical significance is stated on plots (p < 0.05, Mann-Whitney test).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Gene Expression, Control, Biomarker Discovery, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Expression of Mid-1 over the course of AIA. (A) Expression of Mid1 RNA in the knee joints of wt and Fas −/− mice with arthritis, assessed by in situ hybridization (RNAScope). Arrows, brown signal resulting from binding of Mid1 probe, bars, 50 µm. Slides for RNA Scope were selected from a representative experiment used for histology and µCT analysis. Three joints from wt and Fas −/− mice with arthritis were hybridized, with similar results, and representative images are shown. (B) Western blot analysis of Mid-1 expression in knee tissue extracts of wt and Fas −/− AIA mice, and analysis of Mid-1 signal intensity, normalized to total protein signal intensity, acquired from membranes using stain-free technology . Protein extraction was performed from 6 wt and 2 Fas−/− knees harvested from one experiment. (C) Expression of Mid1 in various tissues of wt (n = 2) and Fas −/− mice (n = 2) with AIA. Tissues were harvested from a single experiment. (D) Expression of Mid1 and IL-1β in total joint tissue extracts of wt mice during immunization (d3-21), early (d24), and 1 week after arthritis induction (d28) in a single experiment. Gene expression was determined by RT-PCR and normalized to the expression of β-actin , n = 3, markers, individual values, horizontal lines, median (IQR), * denotes p < 0.05, in comparison to NI group (Kruskal-Wallis test).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Expressing, In Situ Hybridization, RNAscope, Binding Assay, Western Blot, Staining, Protein Extraction, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Myeloid-specific ablation of Fas does not ameliorate antigen-induced arthritis (AIA). Fas fl/fl LysMCre +/− were crossed with Fas fl/fl mice to produce littermates with conditional ablation of Fas in myeloid cells (Fas fl/fl LysMCre +/− , Cre + ) and controls (Fas fl/fl LysMCre −/− , Cre – ). Experiments were repeated 3 times and data are cumulative of minimum 2 representative experiments. (A) Bone marrow cells from Cre − , Cre + and Fas −/− mice were stained with anti-Fas(CD95)-AF488 or corresponding isotype control, and CD11b-PE, Gr-1-PECy7, F4/80-APCCy7, and CD3/B220/NK1.1-APC antibodies. Mean fluorescence intensities (MFI) for each population are shown as individual values (markers), horizontal lines and bars are mean ± SD. Mice were sacrificed on a day 10 post-i.a. injection and arthritis was assessed by (B) measuring knee diameters and semi quantitative visual soring using appropriate scale (0-no arthritis, 1-discrete localized thickening of the joint capsule, 2-mild swelling, absence of sharp patellar ligament contour, 3-clear swelling with diffuse thickening of the joint capsule, 4-severe swelling and deformity, visible through the skin). (C) Subchondral epiphyseal bone volume was assessed by µCT in Cre-NI − (n = 7 4m/3f), Cre − AIA (n = 13, 5m/8f), Cre + NI (n = 6, 4m/2f), Cre + AIA (n = 14, 10m/4f). The following variables were analyzed in distal femoral epiphyses: trabecular bone volume (BV/TV, %), trabecular number (Tb.N., mm -1 ), trabecular thickness (Tb.Th., µm), and trabecular separation (Tb.Sep., µm). (D) Expression of Fas, TNF, IL-1β and Mid1 mRNA in total knee joint tissue from Cre − mice with AIA (Cre − , n = 4, 2m/2f), and Cre + mice with AIA (Cre + , n = 6, 4m/2f). Markers represent individual values, horizontal lines and error bars are mean ± SD (A, B top panel, C) or median (IQR) (B bottom panel, D); statistical significance is marked on plots with red lines connecting experimental groups with p < 0.05 (ANOVA and Student-Newman-Keuls post hoc test, A, B top panel, C; Kruskal-Wallis test, B bottom panel; Mann-Whitney test, D).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Staining, Control, Fluorescence, Injection, Expressing, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Proinflammatory and apoptotic effect of anti-Fas antibody on cells of myeloid (CD11b + Gr1 + ) lineage. Inflammation was induced by 2 μg/mL heat-inactivated Mycobacterium tuberculosis , and after incubation for 1 h/37°C, appropriate combinations of anti-Fas antibody, isotype control antibody, and protein G were added. After incubation for 18h/37°C cells were harvested for flow cytometry and/or RNA isolation. (A) Proportion of apoptotic (annexin V + ) cells, and expression of proinflammatory IL-1β and Mid1 in groups of non-stimulated (NS) and inflammatory-stimulated cells (S) treated with 1 μg/ml isotype control antibody (NI IgG) and/or 1 μg/ml protein G (Prot G) and/or 1 μg/ml anti-Fas antibody (aFas), (B) Expression of proinflammatory cytokines in groups of non-stimulated (NS) and inflammatory-stimulated cells (S) treated with low doses (0.01–0.1 μg/ml) of anti-Fas antibody. 10 6 cells were plated per well of a 96-well culture plate. Cells were grown in duplicates (n = 2, A) or triplicates (n = 3, B), and experiments were repeated three times with similar results. Markers, individual values, horizontal lines, mean ± SD. Statistically significant difference within groups of S and NS cells is marked with red lines connecting experimental groups with p < 0.05, statistically significant difference between identically treated S and NS cells is marked with blue lines connecting experimental groups with p < 0.05, p values are marked on plots (ANOVA and Student-Newman-Keuls post-hoc test).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Incubation, Control, Flow Cytometry, Isolation, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Effects of Mid-1 inactivation on the expression of pro-inflammatory cytokines in inflammatory-stimulated cells. (A) Pharmacological inhibition of Mid-1 in inflammatory-stimulated bone marrow cells. Cells were pre-treated with 20 µM metformin or 20 µM of Mid-1 peptide antagonist (GSK'364A) for 1h/37°C which was followed by the addition of heat inactivated Mycobacterium tuberculosis in final concentration of 10 µg/µL. After overnight incubation at 37°C, expression of proinflammatory cytokines IL-1β , and TNF and concentration of TNF -α were assessed by RT-PCR and ELISA. Experiments were performed in duplicates to quadruplicates, and values are calculated as ratios of means of expression/concentration of cytokine in the stimulated cells treated with metformin or GSK'364A over means of expression/concentration of cytokine in stimulated untreated cells in each experiment (n = 3). (B) Expression of inflammatory cytokines in inflammatory-stimulated bone marrow cells from wt and Mid1 −/− mice treated with agonistic anti-Fas antibody. Cells were treated with 10 µg/µL heat-inactivated Mycobacterium tuberculosis and 0.1 µg/mL anti-Fas antibody. After overnight incubation at 37°C, expression of proinflammatory cytokines IL-1β , and TNF and concentration of TNF -α were assessed by RT-PCR and ELISA. Experiments were performed in duplicates to quadruplicates, and values are calculated as ratios of means of expression/concentration of cytokine in the stimulated cells treated with metformin or GSK'364A over means of expression/concentration of cytokine in stimulated untreated cells in each experiment (n = 3). Horizontal line, median; boxes, IQR; whiskers, range; p values are marked on plots (A, Kruskal-Wallis test; B, Mann- Whitney test).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Expressing, Inhibition, Concentration Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Midline 1 associated with Fas signaling enhances murine antigen-induced arthritis

doi: 10.3389/fcell.2025.1451093

Figure Lengend Snippet: Mid-1 inactivation moderately alleviates antigen-induced arthritis (AIA). (A) AIA was induced in mice with a gene knockout for Mid1 ( Mid1 −/− ) and wt mice. Visual score, knee diameters and µCT values represent cumulative data from 4 separate experiments, while histology score was assessed in one of the representative experiments. Mice were sacrificed on a day 10 post-i.a. injection and arthritis was assessed by (B) measuring knee diameters and semi quantitative visual soring in wt NI (n = 21), wt AIA (n = 23), Mid1 −/− NI (n = 10), Mid1 −/− AIA (n = 20) using appropriate scale (0-no arthritis, 1-discrete localized thickening of the joint capsule, 2-mild swelling, absence of sharp patellar ligament contour, 3-clear swelling with diffuse thickening of the joint capsule, 4-severe swelling and deformity, visible through the skin). (C) Total histology score in wt NI (n = 6), wt AIA (n = 6), Mid1 −/− NI (n = 4), and Mid1 −/− AIA (n = 9). Scoring was performed according to synovial thickening, presence of exudate in joint space, cartilage degradation, and subchondral bone damage, according to the 3 point scale. (D) Subchondral epiphyseal bone volume was assessed by µCT in wt NI (n = 21), wt AIA (n = 23), Mid1 −/− NI (n = 10), Mid1 −/− AIA (n = 20). The following variables were analyzed in distal femoral epiphyses: trabecular bone volume (BV/TV, %), trabecular number (Tb.N., mm -1 ), trabecular thickness (Tb.Th., µm), and trabecular separation (Tb.Sep., µm). (E) Representative 3D models of distal femora, from µCT reconstruction images. Markers represent individual values, horizontal lines and error bars are mean ± SD (B left panel, C) or median (IQR) (B right panel, D); statistical significance is marked on plots with red lines connecting experimental groups with p < 0.05 (ANOVA and Student-Newman-Keuls post hoc test, B left panel, C; Kruskal-Wallis test, B right panel, D).

Article Snippet: After blocking with 5% dry milk in 0.1% PBST for 1h/RT, membranes were incubated with rabbit anti-Mid1 antibody (1:500, Thermo Fisher Scientific Cat# PA5-36305, RRID:AB_2553463), overnight at 4°C, and then with HRP conjugated anti-rabbit antibody (1:50,000, Jackson ImmunoResearch) for 1h/RT.

Techniques: Gene Knockout, Injection

Journal: Current Biology

Article Title: Systems mapping of bidirectional endosomal transport through the crowded cell

doi: 10.1016/j.cub.2024.08.026

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mGFP, mouse anti-RFP (Chromotek 5F8), rabbit anti-RILP (Q31 ), rabbit anti-FYCO1 (NBP1-47266, Novus), rabbit anti-ZFYVE25 (Protrudin) (sc-102174 Santa Cruz), rabbit anti-Rab5 (C8B1, Cell signaling), rabbit anti-Rab7 (D95F2, Cell signaling), rabbit anti-KIF5B (ab25715, Abcam), mouse anti-DyneinHC (C-5, sc-514579 Santa Cruz), mouse anti-β-Actin (AC-15, Sigma), mouse anti-Lis1 (H-7, Santa Cruz), mouse anti-MAPRE1 (clone 5, BD), rabbit anti-NCKAP5L (PA5-59404, Invitrogen), rabbit anti-MID (NBP1-26612, Novus), mouse anti-p150 glued (clone1, BD) followed by secondary Goat-anti-mouse-HRP (G21040, Invitrogen) or goat-anti-rabbit-HRP (G21234, Invitrogen) were used for detection of endogenously-tagged proteins by Western blot.

Techniques: Recombinant, CRISPR, Sequencing, Introduce, Clone Assay, Software

Journal: Current Biology

Article Title: Systems mapping of bidirectional endosomal transport through the crowded cell

doi: 10.1016/j.cub.2024.08.026

Figure Lengend Snippet: EB1, MID1, and CEP169 recruit Lis1 and dyneinHC, but not p150 glued , to the microtubule growing plus ends (A) EGFP-eLis1 (green) and SPYtubulin (red) localization in MelJuSo cells. Arrows show microtubule plus ends ( A). (B) Lis1-positive spots/μm 2 after indicated MAPs’ depletion compared with control (siC) ( Figure S7 D) ( N = 10–26 cells, n = 2 independent experiments). (C) EGFP-eLis1 (green) and LEs (LysoTracker, blue) localization in relation to eEB1-mScarlet, mScarlet-eCEP169, and mScarlet-eMID1 (red) ( B–S14D). (D) 30-s kymographs of EGFP-eLis1, eEB1-mScarlet, and LysoTracker of area in (C) (dotted line). (E) DyneinHC-positive spots/μm 2 after indicated MAPs’ depletion, compared with control (siC) ( Figure S7 H) ( N = 13–18 cells, n = 2 independent experiments). (F) p150 glued -positive spots/μm 2 after indicated MAPs’ depletion, compared with control (siC) ( Figure S7 J) ( N = 14–15 cells, n = 2 independent experiments). (G) HA-RILP (unstained) overexpression in mScarlet-eDyneinHC, EGFP-Lis1, or EGFP-ep150 glued cells (green), fixed and antibody-stained for CD63 (red). (H) Manders quantification of data in (G) ( N = 14–21 cells, n = 2 independent experiments). (I) EGFP-eLis1 (green) and mScarlet-eRab5a, mScarlet-eRab6a, or mScarlet-Rab7a (red) in time . (J) Distance quantification of data in (I). Measured is the shortest distance from detected vesicles to the Lis1-positive mask. Percentage of endosomes with distance <180 nm is plotted ( N = 20–31 cells, n = 3 independent experiments). Plots report mean; error bars reflect ± SD. t test or one-way ANOVA, ∗∗∗ p < 0.001, ∗∗ p < 0.005; ns, not significant. Scale bars as indicated. See also Figure S7 and and .

Article Snippet: Rabbit polyclonal anti-MID1 , Novus , Cat#NBP1-26612; RRID: AB_1853386.

Techniques: Control, Over Expression, Staining

Journal: Current Biology

Article Title: Systems mapping of bidirectional endosomal transport through the crowded cell

doi: 10.1016/j.cub.2024.08.026

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MID1 , Novus , Cat#NBP1-26612; RRID: AB_1853386.

Techniques: Recombinant, CRISPR, Sequencing, Introduce, Clone Assay, Software

Journal: Current Biology

Article Title: Systems mapping of bidirectional endosomal transport through the crowded cell

doi: 10.1016/j.cub.2024.08.026

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MID1 , Novus , Cat#NBP1-26612; RRID: AB_1853386.

Techniques: