anti mid1  (Boster Bio)

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: 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: 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:

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: (A) Schematic depicting the MID1 gene including exons. Numbers indicate base pairs. Dashed lines indicate reported mutations in the MID1 gene . Protein structure including major domains and amino acids. Below the MID1 protein structure, the frequency of deletions along the gene is summarized. The lowest panel indicates the rare exome variant ensemble learner (REVEL) score along the MID1 gene body. Note the peak of the REVEL score in the N-terminal end of MID1, correlating with the absence of reported pathogenic variants in patients. (B) Scheme highlighting the experimental procedure to derive fibroblasts from a male healthy donor and perform CRISPR/Cas9-mediated perturbations in coding exon 1 of MID1 in the hiPSCs derived from these fibroblasts. The resulting Rm1 and Rm2 mutations are caused by a 1-bp insertion and a 2-bp deletion, respectively. Besides the full-length MID1 protein that is produced when translation starts from ATG1, further alternative ATGs and their expected usage in the different MID1 hiPSC lines are schematized. (C) Quantitative RT–PCR of the expression levels of MID1 using specific primers to detect exons 1–2, 6–7, and 8–9 across Ctrl, Rm1, and Rm2 hiPSC lines (n = 4). (D) Western blot showing MID1 protein expression using an antibody against the C-terminus of MID1. Actin is used as a loading control. The arrow indicates full-length MID1, whereas # indicates truncated MID1 proteins of 69, 64, and 58/57 kD, and the star indicates an unspecific band. (E) Scheme depicting full-length and N-terminally truncated MID1 proteins resulting from the usage of alternative ATGs. The lowest scheme depicts the gene structure of a patient-derived MID1 variant exhibiting a 4-bp deletion at the C-terminal end. The color code on the left indicates lines in which this isoform is present (turquois: Ctrl, blue: Rm1, purple: Rm2, black: del4). (E, F) Micrographs showing cellular co-localization of MID1 isoforms fused to GFP with TUBB3 (orange) after overexpression of MID1-GFP constructs (summarized in (E)) in HeLa cells. The color of the box above (upper panel) or below (lower panel) the pictures indicates presence of this isoform in the respective hiPSC lines (turquois: Ctrl, blue: Rm1, purple: Rm2, black: del4). Note the formation of aggregates and loss of microtubule association upon overexpression of MID1 with a 4-bp deletion in the C-terminus as shown previously . Scale bar = 15 μm. (G) Images of representative Ctrl, Rm1, and Rm2 d30 brain organoid slices show the cellular organization through DAPI staining. The yellow dashed lines highlight representative ventricular zone-like structures (VZLS). (H) Quantification of the areas of VZLS covering the total area of brain organoid slices shown as box plots with jitters indicating individual d30 organoids. The data reveal a decrease in the contribution of VZLS to the brain organoids in the MID1 Rm organoids. Dots represent individual organoids derived from different batches, as indicated by distinct colors. Ctrl: n = 29 from seven batches, Rm1: n = 12 from four batches, Rm2: n = 12 from four batches, KO: n = 14 from three to six batches. Mann-Whitney- U test. ** P < 0.01, *** P < 0.001. Exact P -values (top to down) 0.0017, 6.5 × 10 −7 , 0.0027. Boxplots show median, quartiles (box), and range (whiskers). (I) Immunofluorescence stainings show the expression of MID1 (green) and PAX6 (magenta) in d30 brain organoid slices. MID1 signal can be detected in Ctrl and Rm1, but not in Rm2 or KO brain organoids. For (F, G), DAPI was used to counterstain nuclei. For (G, I), scale bar = 100 μm.

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Variant Assay, CRISPR, Derivative Assay, Produced, Quantitative RT-PCR, Expressing, Western Blot, Control, Over Expression, Construct, Staining, MANN-WHITNEY, Immunofluorescence

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: (A) Electropherograms show sequencing results of genome-edited hiPSC lines. Highlighted are the 2-bp deletion in Rm1 (upper panel) and the 1-bp insertion within MID1 exon1 in the Rm2 hiPSC line (lower panel). (B) Scheme depicting the experimental approach to generate a MID1 full knockout by CRISPR/Cas9 in an isogenic male hiPSC line. (C) Bright field images of d3 embryoid bodies generated from Ctrl, Rm1, Rm2, and MID1 KO hiPSC lines. Scale bar = 200 μm. (D) Quantification of embryoid body size (d3) shown as fold change over Ctrl embryoid bodies. n = 52 from five batches (Ctrl), n = 32 from three batches (Rm1, Rm2), n = 20 from two batches (KO). Mann-Whitney- U tests were performed. Boxplots show median, quartiles (box), and range (whiskers). *** P < 0.001. Exact P -values (top to down) 5.4 × 10 −8 , 9.1 × 10 −12 , 3.8 × 10 −14 . (E) Images show immunofluorescence stainings against activated Caspase3 (actCASP3) to detect apoptotic cells in d30 organoids of all conditions. DAPI was used to counterstain nuclei. Scale bar = 500 μm. (F) Immunofluorescent staining of a 2-mo organoid derived from WT pluripotent stem cells showing expression of MID1 (green) and PAX6 (magenta). Scale bar = 500 μm.

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Sequencing, Knock-Out, CRISPR, Generated, MANN-WHITNEY, Immunofluorescence, Staining, Derivative Assay, Expressing

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: (A) Immunofluorescence stainings of brain organoid slices showing SOX2 (green) and MAP2 (magenta) positive cells in d30 Ctrl, Rm1, Rm2, and KO organoids. DAPI was used to counterstain nuclei. Scale bar = 500 μm. (B) Quantification of the relative contribution of neural areas as quantified by the fraction of SOX2+ and MAP2+ per total area in d30 organoids reveals a decrease in the MID1 Rm organoids compared with Ctrl and KO organoids (Rm = Rm1 + Rm2), as shown by violin ad jitter plots. Mann-Whitney- U Test. Exact P -values (top to down) 0.01, 0.0017, 0.057. (C) Within the neural area, quantification of MAP2 in d30 organoids revealed reduced neural differentiation in the MID1 Rm organoids, as shown by violin and jitter plots. Mann-Whitney- U test. Exact P -values (top to down) 0.02, 0.11, 0.15. For (B, C), * P < 0.05, ** P < 0.01, ns, not significant. Dots represent individual organoids. Ctrl: n = 26 from six independent batches, Rm: n = 14 from six independent batches, KO: n = 16 from three independent batches. (D) Principal component analysis segregated the transcriptomes of the experimental hiPSC as highlighted by 95% confidence ellipses (dashed lines). (E) The PC plot depicts the transcriptional divergences of the Ctrl, Rm1, and Rm2 hiPSC lines from the hiPSC state throughout early differentiation into brain organoids (d5, d8, d11 of differentiation). Confidence ellipses (dashed lines) illustrate that Rm lines cluster together but differ from Ctrls. (F) Euler diagram showing the number of genes upregulated during differentiation from hiPSC to d5/d8/d11 in each condition and comparing the different conditions. (G) The Euler diagram showing the number of downregulated genes across experimental conditions during early neural differentiation. (F, H) Bar graph showing the top 20 GO terms significantly enriched in the genes upregulated specifically in the Ctrl organoids (i.e., 162 genes from (F)). (F, I) Top 20 GO terms enriched upon analysis of the Rm-specific upregulated genes, i.e., 97 genes from (F).

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Immunofluorescence, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: (A) Scheme highlighting the experimental outline of the bulk RNA-sequencing experiments. (B) Heatmap depicting the normalized counts per million (CPM) values of MID1 , MID2 , POU5F1 (OCT4), SOX2 , NANOG , and SALL4 across sampled human-induced pluripotent stem cell lines. (C) Heatmaps depicting the scaled normalized values of MID1 across conditions and time points. (D) Analysis of the genes down-regulated specifically in the Ctrl organoids (i.e., 78 genes from ). The top 20 significantly enriched GO terms are shown. (E) Top 20 significantly enriched GO terms emerging from the analysis of Rm specifically down-regulated genes, that is, 87 genes from . (F) Heatmap depicting the scaled normalized CPM values of HOXB4 and HOXB5 across conditions and time points indicating caudalization in Rm samples. (G) Heatmaps depicting the scaled normalized CPM values of DLX5 across conditions and time points showing incapacity to induce ventral genes.

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: RNA Sequencing Assay

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: (A) Box and jitter plots show the fold change of the expression of patterning genes BMP6 , LMX1A , OTX2 , TTR , and PTCH1 normalized to GAPDH in d30 Ctrl, Rm1, Rm2, and MID1 KO organoids. Mann-Whitney- U test, * P < 0.05, ** P < 0.01, *** P < 0.001. ns, not significant. Exact P -values (left to right) 0.0083, 0.014, 0.34, 0.037, 0.0096, 0.49, 0.084, 0.0014, 0.8, 0.0003, 0.00004, 0.29, 0.0053, 0.076, 0.017. ( BMP6 : Ctrl: n = 15, six batches; Rm1: n = 9, three batches; Rm2: n = 7, three batches; KO: n = 6, three batches. LMX1A , OTX2 , TTR : Ctrl: n = 16, six batches; Rm1: n = 9, three batches; Rm2: n = 9, three batches; KO: n = 6, three batches; PTCH1 : Ctrl: n = 16, six batches; Rm1: n = 9, three batches; Rm2: n = 7, three batches, KO: n = 6, three batches). (B) Brain organoids (d30) were used to quantify mRNA expression levels of TTR and ASCL1 across conditions. Note the increase in TTR expression at the expense of the ventral marker ASCL1 in the MID1 Rm mutant organoids. (Ctrl: n = 9, three batches; Rm1: n = 7, three batches; Rm2: n = 9, three batches). (C) Correlation matrix of the logarithmic fold change values of MID1 mutants versus Ctrl shows coregulated nodes and overall anti-correlation of dorsal choroid plexus marker TTR with the patterning genes PTCH1 and GLI1 . ( BMP6 : Ctrl: n = 15, six batches; Rm1: n = 9, three batches; Rm2: n = 7, three batches; LMX1A , OTX2 , TTR , DLX2 : Ctrl: n = 16, six batches; Rm1: n = 9, three batches; Rm2: n = 9, three batches; PTCH1 : Ctrl: n = 16, six batches; Rm1: n = 9, three batches; Rm2: n = 7, three batches; GLI1 : Ctrl: n = 16, six batches; Rm1; n = 9, three batches; Rm2; n = 8, three batches; FOXA2 : Ctrl: n = 9, three batches; Rm1; n = 9, three batches; Rm2; n = 9, three batches; ASCL1 : Ctrl: n = 9, three batches; Rm1; n = 8, three batches; Rm2; n = 9, three batches). (D) Line-plot showing the temporal pattern of GBX2 misexpression (ln of FC) along early differentiation in different conditions; solid line: mean; shade: 95% confidence interval. Note the constant increase in GBX2 in the Rm lines, contrasting the expression in the KO line (Ctrl: n = 6 from two batches, Rm1, Rm2, KO: n = 3 from one batch). (E) Box and jitter plots show the natural logarithm of the fold change values versus mean of Ctrl of ATOH1 expression normalized to GAPDH in d30 organoids across conditions. (Ctrl: n = 6 from two batches, Rm1, Rm2, KO: n = 3 from one batch). Mann-Whitney- U test, * P < 0.05, ns, not significant. Exact P -values (top to down) 1, 0.024, 0.024. (F) Images show sections of d30 organoids of all conditions (Ctrl, Rm1, Rm2, KO) stained for TTR. DAPI was used to counterstain nuclei and visualize cellular organization. Orange dashed line indicates zoom-in areas shown in the lower panel. Note that the tissue positive for TTR is organized as monolayered epithelium. (G) Quantification of TTR-positive areas covering the area of organoid indicated as fold change over Ctrl organoids across conditions. Different batches are indicated by distinct colors. Ctrl: n = 15 from six batches, Rm1, Rm2: n = 7 from four batches, KO: n = 9 from three batches. Mann-Whitney- U test. *** P < 0.001; ns, not significant. Exact P -values (top to down) 0.77, 0.00032, 0.00092. For (A, B, D, E, G), dots represent individual organoids. Boxplots show median, quartiles (box), and range (whiskers). (H) Micrographs show immunofluorescence stainings using an antibody against the cilia marker ARL13B in d30 organoids. Orange dashed box highlights insets magnified in the lower panel. Arrow heads point towards multiciliated cells. (I) SOX9 protein expression in Ctrl, Rm1, Rm2, and MID1 KO d30 organoids. Note the high expression in ventricular zone-like structures and the choroid plexus-like areas. For (F, H, I), DAPI was used to counterstain nuclei. (F, H, I) Scale bars = 500 μm (F, H upper, I), 100 μm ((F), lower), 25 μm ((H), lower).

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Expressing, MANN-WHITNEY, Marker, Mutagenesis, Staining, Immunofluorescence

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: gRNAs used in this study.

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Sequencing

Journal: Life Science Alliance

Article Title: Absence of the RING domain in MID1 results in patterning defects in the developing human brain

doi: 10.26508/lsa.202302288

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Techniques: Sequencing