Journal: Disease Models & Mechanisms

Article Title: Genetic mutations linked to Parkinson's disease differentially control nucleolar activity in pre-symptomatic mouse models

doi: 10.1242/dmm.028092

Figure Lengend Snippet: hA53T-SNCA is expressed in DA neurons in the cytoplasm, the nucleus and the nucleolus. Representative confocal microscopy images of paraffin sections immunostained with TH (green), human SNCA (α-syn; red)- and NCL (blue)-specific antibodies from 3-month-old wild-type (A-C) and hA53T-SNCA-overexpressing transgenic mice in a PINK1 KO background (hA53T-SNCA/PINK1KO) (D-F). B,C and E,F show higher magnifications of the region indicated by the arrows in A and D, respectively. Scale bar: 25 μm (A,D) and 12 µm (B-F).

Article Snippet: The following TaqMan gene expression assays were used: TIF-IA (Mm01344420_m1), nucleolin (Mm01290591_m1), nucleophosmin (Mm02391781_g1), and Tbp (TATA-binding protein) (Mm00446973-m1) (Applied Biosystems/Life Technologies).

Techniques: Confocal Microscopy, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Genetic mutations linked to Parkinson's disease differentially control nucleolar activity in pre-symptomatic mouse models

doi: 10.1242/dmm.028092

Figure Lengend Snippet: Nucleolar number is altered in hA53T-SNCA/PINK1KO mice at pre-symptomatic stages. (A) Quantification of nucleoli detected by ISH with 47S-specific riboprobe in TH-positive neurons of 3-month-old wild-type and hA53T-SNCA/PINK1KO mice ( N =4) in SN (left) and VTA (right) expressed as percentage of TH-positive neurons with 1, 2-3 or no nucleoli (nuc). (B) Representative images of ventral midbrain paraffin sections from 3-month-old wild-type and hA53T-SNCA/PINK1KO mice immunostained with NCL (brown)- and TH (blue)-specific antibodies. (C,D) Quantification of the number of nucleoli detected by NCL (C) and NPM (D) immunostaining in TH-positive neurons of wild-type and hA53T-SNCA/PINK1KO mice in SN (top) and VTA (bottom) expressed as percentage of TH-positive neurons with 1, 2-3 or no nucleoli ( N =4 mice). (E) Analysis of Ncl (top) and Npm (bottom) mRNA levels by qRT-PCR at 3, 16 and 18 months in wild-type ( N =5,3,3) and hA53T-SNCA/PINK1KO mice ( N =6,3,3) expressed as fold change to respective controls and normalized by Tbp . All data are mean±s.e.m.; * P <0.05; ** P <0.01; *** P <0.001 in comparison to wild-type, as determined by two-way ANOVA followed by Sidak's multiple comparison test. Scale bar: 50 μm.

Article Snippet: The following TaqMan gene expression assays were used: TIF-IA (Mm01344420_m1), nucleolin (Mm01290591_m1), nucleophosmin (Mm02391781_g1), and Tbp (TATA-binding protein) (Mm00446973-m1) (Applied Biosystems/Life Technologies).

Techniques: Immunostaining, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Genetic mutations linked to Parkinson's disease differentially control nucleolar activity in pre-symptomatic mouse models

doi: 10.1242/dmm.028092

Figure Lengend Snippet: rDNA transcription is not reduced in PINK1 KO or DJ-1/PINK1 DKO mice. (A) Analysis of pre-rRNA (1-130) and pre-rRNA (597-765) by qRT-PCR in controls ( N =3), PINK1 KO ( N =8) and DJ-1/PINK1 DKO mice ( N =8) expressed as fold change to respective controls normalized by Gapdh . (B) Representative images of ISH with a 47S-specific riboprobe (blue) in combination with immunohistochemistry with a TH-specific antibody (brown) in 9-month-old control, PINK1 KO and DKO mice. (C) Quantification of the area occupied by the 47S staining in control, DJ-1KO, PINK1 KO and DKO mice expressed as fold change relative to respective controls in SN and VTA ( N =3 mice). (D) Analysis of Ncl , Npm and TIF-IA mRNA levels by qRT-PCR in wild-type ( N =3), PINK1 KO ( N =8) and DJ-1/PINK1 DKO ( N =8) mice at 9 months expressed as fold change to respective controls normalized by Tbp . All data are mean±s.e.m. * P <0.05 as determined by one-way ANOVA followed by Tukey's multiple comparisons test. Scale bar: 50 μm.

Article Snippet: The following TaqMan gene expression assays were used: TIF-IA (Mm01344420_m1), nucleolin (Mm01290591_m1), nucleophosmin (Mm02391781_g1), and Tbp (TATA-binding protein) (Mm00446973-m1) (Applied Biosystems/Life Technologies).

Techniques: Quantitative RT-PCR, Immunohistochemistry, Staining

Journal: Disease Models & Mechanisms

Article Title: Genetic mutations linked to Parkinson's disease differentially control nucleolar activity in pre-symptomatic mouse models

doi: 10.1242/dmm.028092

Figure Lengend Snippet: Loss of DJ-1 and PINK1 does not exacerbate the toxic effects of nucleolar stress in DA neurons. (A) Experimental design. (B) Representative confocal images showing nucleolar integrity in DA neurons by immunofluorescence staining with NCL (red) and TH (green) antibodies in control, DJ-1/PINK1 DKO, TIF-IA conditional mutants (cKO) and TKO mice. (C) Representative IHC images of p53 protein (brown) in TH-positive neurons (blue) in control and TKO mice 7 weeks after TAM injections. Neurons showing increased p53 levels are indicated by arrowheads. (D) Representative images of striatal TH immunoreactivity on vibratome sections of control, DJ-1/PINK1 DKO, TIF-IA conditional mutants (cKO) and TKO mice. (E) Quantification of TH immunoreactivity in the striata of control ( N =5), DKO ( N =3), cKO ( N =4) and TKO ( N =5) mice. (F) Analysis of dopamine content by HPLC-ED in the striata of control ( N =13), DKO ( N =9), cKO ( N =4) and TKO ( N =5) mice. Data are mean±s.e.m.; * P <0.05; ** P <0.01; *** P <0.001 as determined by one-way ANOVA followed by Tukey's multiple comparison test. ww, wet weight; TAM, tamoxifen. Scale bar: 30 μm (B), 50 μm (C) and 200 μm (D).

Article Snippet: The following TaqMan gene expression assays were used: TIF-IA (Mm01344420_m1), nucleolin (Mm01290591_m1), nucleophosmin (Mm02391781_g1), and Tbp (TATA-binding protein) (Mm00446973-m1) (Applied Biosystems/Life Technologies).

Techniques: Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: ARID1a-DNA Interactions Are Required for Promoter Occupancy by SWI/SNF

doi: 10.1128/MCB.01008-12

Figure Lengend Snippet: The ARID domain of ARID1a is required for BAF-A occupancy at THBS1. (A) Mouse/human VISTA alignment of the THBS1 promoter and 5′ transcribed region. Salmon-colored peaks denote evolutionarily conserved regions, whereas lavender peaks denote exons. The locations of ChIP amplicons within this interval are plotted. (B to H) Formaldehyde-cross-linked chromatin from wild-type and Arid1aV1068G/V1068G MEFs was immunoprecipitated with ARID1a, ARID1b, ARID2, BRG1, BRM, INI1/SNF5, or Pol II antibodies. DNA was amplified by quantitative PCR to determine if loss of ARID-DNA binding leads to changes in BAF-A occupancy across THBS1. An intergenic, nonconserved downstream region (located at approximately kb +20) and two unlinked promoter control elements (GAPDH and INS-1) were used as negative genomic controls. Data were plotted as the percentage of total input or chromatin bound. (I) Whole-embryo protein lysates from triplicate pooled samples were used to examine THBS1 protein (reduced, monomeric form) expression differences by Western blotting. An overexposed image of the Western blot was also included to further emphasize these expression differences. The constitutive nuclear matrix protein, nucleolin, was used as a loading control. (J) cDNA synthesized from RNA isolated from wild-type or Arid1aV1068G/V1068G MEFs was used in a quantitative PCR to examine THBS1 expression differences following transfection with mock (nontargeting control), BRG1, or BRM siRNA. (K) Normalized luciferase activity of the THBS1 −2.8 kb promoter-luciferase fragment cotransfected with 0.05 to 0.5 μg of wild-type or mutant HA-mARID1a expression plasmids into NIH 3T3 cells. Cells cotransfected with the empty luciferase vector (−luc) or THBS1 −2.8 kb promoter-luciferase fragment and with empty pcDNA expression plasmids served as negative and positive controls, respectively. (L) Normalized luciferase activity of THBS1 −2.8 kb and −0.48 kb promoter-luciferase reporter plasmids cotransfected with 0.25 μg of pcDNA only (−) or wild-type or mutant HA-mARID1a expression plasmids. Empty luciferase reporter plasmid was used as a negative control. (M) Summary model of ChIP and expression data. Error bars in panels B to H and in panel J represent the SEMs. Error bars in panels K and L represent the standard deviations. Significant differences were calculated using a two-tailed Student t test (*, P < 0.05).

Article Snippet: Western blotting was performed using standard procedures and the following antibodies: ARID1a (A301-040A or A301-041A; Bethyl Labs), ARID1b (sc-32762; Santa Cruz), ARID2 (A302-230A; Bethyl Labs), BRG1 (sc-17796; Santa Cruz), BRM (sc-6450; Santa Cruz), INI1/SNF5 (A301-087A; Bethyl Labs), BAF60a (611728; BD Biosciences), cullin-2 (ab1870; Abcam), THBS-1 (MS-421-P0; Thermo Scientific), hemagglutinin (HA) tag (A190-108A; Bethyl Labs), β-actin (ab8226; Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma), and nucleolin (A300-711A; Bethyl Labs).

Techniques: Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Control, Expressing, Western Blot, Synthesized, Isolation, Transfection, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control, Two Tailed Test

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 1. Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3. A, schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type b-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B, immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 (magenta). EGFP (green). DAPI (blue). C, western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 (upper panel) and anti-AIS1 (lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP- CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D, schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 40,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 2. Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody. A, immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 (green) and CF555-conjugated anti-CAPN3 (red) antibody. The scale bar represents 20 mm. B, scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C, mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 14 cells. Each plot represents an individual cell value. Mean ± SD. ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 3. Ouabain Ca2+ dependently activates CAPN3:S606L in HeLa cells. A, immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 (green) and anti-CAPN3 (magenta) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 mm. B, change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ***p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C, Ca2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca2+ signals of HeLa cells upon 10 mM ATP stimulation are also indicated. D, ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ***p = 2.45 x 10-9, Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 mM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E, pretreatment with a Ca2+ chelator BAPTA- AM (25 mM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and b−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F, increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. **p = 0.01445, ***p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 4. Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles. A, autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B, changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A. Mean ± SD. N = 34. *p < 0.05, ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. C, validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green: CAPN3; magenta: Actinin (Z-band). The scale bar represents 10 mm. D, intensity profiles of CAPN3 and actinin signals on white bars (2 mm thick) in the panels of C. Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E, immunohis- tochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 (green) and anti-actinin (magenta, Z-band) antibody. The scale bar represents 20 mm. F, intensity profiles of CAPN3 and actinin signals on white bars (2 mm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 5. Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation. A and B, immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before (A) and aftr 1 mM ouabain stimulation for 60 min (B). AIS1 (green), CAPN3 (magenta), and DAPI (blue). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 mm. C, ouabain-induced cytosolic Ca2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D, time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. *p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 40,6-diamidino-2-phenylindole.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of biological chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Figure 7. Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation. A, time-dependent increase in the spectrin fragment (150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment (upper panel). The same membrane was probed with antibodies for GAPDH (lower panel). B, relative band intensities of the 150 kDa spectrin fragments in (A). Mean ± SD. Number of experiments were 3 4. **p < 0.01, one- way ANOVA with Dunnett’s multiple comparison test. C, time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin (upper panel) and GAPDH (lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D, relative intensity of the cleaved band (250 kDa) of talin. Mean ± SD. Number of experiments were 4 6. *p < 0.05, Steel-Dwass test. E, expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported (59). Top and bottom indicated the top and bottom of the gel, respectively. F, immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH (lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma- Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 C overnight.

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell communication networks. (A) Incoming communication patterns of target cells, showing pathways to which each cell type responds. (B) Outgoing communication patterns of secreting cells, illustrating the pathways through which cells send signals, MIF, MK and CXCL pathway exhibit high activity. (C) Network diagram showing the strength of intercellular communication, with connections between various cell types. (D) Scatter plot comparing outgoing and incoming communication strengths across cell populations, with bubble size indicating the number of interactions, malignant cells have higher strength of intercellular communication. (E) Chord diagram depicting communication via the MK pathway between different cell types. (F) Ligand-receptor interaction probabilities within the MK pathway between malignant and other cell types. Dot size represents significance (P-value), and color represents communication probability highlighting the MDK-NCL signaling pathway. (G) Violin plots of MK pathway gene expression levels across cell types, showing gene activity variations, MDK has advancer expression level in malignant cells.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Activity Assay, Gene Expression, Expressing

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Spatial transcriptomics and MDK-NCL signal communication. (A) Niche clustering in spatial transcriptomics samples, identifying distinct ecological zones. (B) Spatial expression of representative markers in key regions: MUC1 (tumor region), LYZ (immune region), COL14A1 (stromal region), and SFTPC (normal region). (C) Violin plots displaying the expression of MUC1, LYZ, COL14A1, and SFTPC across different niches. (D) MCPcounter analysis showing the infiltration of six cell types (e.g., endothelial cells, fibroblasts, immune lineages) across spatial regions. (E) Spatial niche classification, distinguishing tumor, immune-stromal, and normal regions. (F) MDK-NCL ligand-receptor interaction analysis, spatially mapping MDK ligands, NCL receptors, and their binding regions.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell pseudotime analysis. (A) Pseudotime trajectory analysis showing the 6 differentiation states of cells. (B) Subtype classification of malignant cells along the pseudotime trajectory. (C) Pseudotime scores mapped along the differentiation trajectory. (D) UMAP plot visualizing pseudotime scores across individual cells. (E) Box plots comparing pseudotime scores across different malignant cell clusters, cluster 0, 1, and 5 had higher pseudotime scores. (F) UMAP plot of differentiation states, with colors representing distinct states. (G) Stacked bar plots showing the proportion of differentiation states within each malignant cell cluster, cluster 0, 1, and 5 have larger proportion of state 6. (H) Expression dynamics of MK pathway genes (e.g., MDK, NCL, ITG genes) along the pseudotime trajectory, highlighting gene expression changes during differentiation, MDK and NCL express more in the later time.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with the immune microenvironment. (A) Boxplot shows the expression levels of MDK and NCL genes in tumor and control groups, it exhibit higher activity in tumor group. (B) MDK-NCL enrichment scores in tumor and control groups. (C) Relative mRNA expression levels of MDK and NCL in tumor and control groups from in-house data. (D) Relative protein expression levels of MDK and NCL in tumor and control groups from in-house data. (E) Comparison of MDK protein expression levels between tumor and control groups. (F) Comparison of NCL protein expression levels between tumor and control groups. (G) Correlation of MDK and NCL expression with ImmuneScore, StromalScore, ESTIMATEScore, and TumorPurity. (H) Scatter plots depicting the relationship between MDK and NCL expression and immune-related scores (ImmuneScore, StromalScore, ESTIMATEScore) as well as TumorPurity. (I) Comparison of immune cell infiltration scores across high and low MDK-NCL expression groups for 28 immune cell types. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Control, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with immunotherapy response. (A) Comparison of tumor mutation burden (TMB) between high and low MDK-NCL expression groups. (B) Comparison of microsatellite instability (MSI) between high and low MDK-NCL groups. (C) Comparison of dysfunction scores between high and low MDK-NCL groups. (D) Comparison of exclusion scores between high and low MDK-NCL groups. (E) Expression of immunogenic cell death (ICD)-related genes in high and low MDK-NCL groups. (F) Expression levels of CTLA4 and PD1 in high and low MDK-NCL groups. (G) Comparison of immune checkpoint gene expression between high and low MDK-NCL expression groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Comparison, Mutagenesis, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: ( A ) Phosphorylated LIX1L was immunoprecipitated from the cytosolic and nuclear fractions of HEK-293FLG and HEK-293FLG-LIX1L cells using a FLAG antibody. Immunoprecipitates were analyzed through western blot analysis with a LIX1L antibody and phosphorylated serine-, threonine- and tyrosine-specific antibodies (upper panels). In the cytosolic and nuclear fractions of the HEK-293FLG and HEK-293FLG-LIX1L cells treated with 25 μM PY95 as a negative control or PY136, immunoprecipitates obtained using the FLAG antibody were analyzed through a western blot analysis with the LIX1L antibody and phosphorylated tyrosine-specific antibodies (bottom panels). Representative blots from HEK-293FLG and HEK-293FLG-LIX1L cell lines are shown. ( B ) The cell counts of HEK-293FLG and HEK-293FLG-LIX1L cells after treatment with PY136 (left panel). The HEK-293FLG and HEK-293FLG-LIX1L cells were cultured in semisolid methylcellulose media. The HEK-293FLG-LIX1L cells were left untreated or were treated with PY136 (25 μM). After 14 days in culture, colony formation was analyzed, and the cells were viewed using phase-contrast microscopy. The colonies formed from each cell type (3 × 10 2 to 5 × 10 2 cells/plate) were counted following plating onto semisolid methylcellulose media (right upper panel). Original magnification 4x (right bottom panels). These data are shown as the mean ± SD for independent experiments. **p < 0.05, *p < 0.01 . ( C ) The results of the immunoblot analysis of the cytosolic fraction treated with or without RNase in HEK-293FLG-LIX1L cells. The black arrow indicates the FLAG-LIX1L fusion protein. The red arrows indicate the detected proteins associated with the LIX1L-RNA complex. ( D ) Western blot analysis revealed that LIX1L interacted with the RIOK1, nucleolin and PABPC4 proteins in the cytoplasm of HEK-293 cells. In ( A ) and ( D ), the cropped blots were run under the same experimental condition.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Cell Culture, Microscopy

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: The identification of LIX1L-associated proteins by MALDI-TOF/TOF mass spectrometry.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques:

Journal: Brain Pathology

Article Title: Fatal familial insomnia: mitochondrial and protein synthesis machinery decline in the mediodorsal thalamus

doi: 10.1111/bpa.12408

Figure Lengend Snippet: Abbreviated names of genes, their full names, and TaqMan probe references used for the study of mRNA expression of mitochondria, protein synthesis and purine metabolism enzymes including GUS‐β and XPNPEP1 used for normalization.

Article Snippet: NCL , Nucleolin , Hs01066668_m1.

Techniques: Expressing, Binding Assay, Membrane

Journal: Brain Pathology

Article Title: Fatal familial insomnia: mitochondrial and protein synthesis machinery decline in the mediodorsal thalamus

doi: 10.1111/bpa.12408

Figure Lengend Snippet: Quantitative data of cells stained with Nissl stain and immunoreactive with antibodies against voltage‐dependent anion channel (VDAC), glial fibrillary acidic protein (GFAP), CD8 (reactive microglia), nucleolin (NCL), nucleoplasmin 3 (NPM3), histone H3K9me2, ATP5H protein and superoxide dismutase 2 (SOD2) in the mediodorsal thalamus in control and FFI cases. Values are expressed as mean values ± SD. Assessed cell types are named in the left column excepting ATP5H and SOD2 immunoreactive cells which are neurons in controls and glia in FFI cases.

Article Snippet: NCL , Nucleolin , Hs01066668_m1.

Techniques: Staining, Control

Journal: Brain Pathology

Article Title: Fatal familial insomnia: mitochondrial and protein synthesis machinery decline in the mediodorsal thalamus

doi: 10.1111/bpa.12408

Figure Lengend Snippet: Nucleolin (NCL) (A, B), NPM3 (C, D), and H3 dimethyl K9 (H3K9me2) (E, F) immunoreactivity is observed in neurons in the mediodorsal thalamus in controls (A, C, E). Weak NCL immunoreactivity is found in the nucleolus whereas strong NCL immunoreactivity is present in the neuronal cytoplasm. NPM3 immunoreactivity is observed in the nucleolus, whereas H3K9me2 immunolabeling is localized in the nucleus. Decreased NCL, NMP3, and H3K9me2 expression is found in FFI cases (B, D, E) due to the marked decrease in the number of neurons. Paraffin sections slightly counterstained with haematoxylin; bar = 25 µm.

Article Snippet: NCL , Nucleolin , Hs01066668_m1.

Techniques: Immunolabeling, Expressing

Journal: Brain Pathology

Article Title: Fatal familial insomnia: mitochondrial and protein synthesis machinery decline in the mediodorsal thalamus

doi: 10.1111/bpa.12408

Figure Lengend Snippet: A. Western blots of nucleolin, initiation (eIF2α, P‐eIF2α, eIF5) and elongation factors (eEF1A, eEF2) in mediodorsal thalamus in FFI and control cases. β‐actin was used to normalize total protein. B. Densitometric analysis shows significant reduction of NCL and eEF1A. Results were analyzed by Graphpad Prism with Student's t‐test when the distribution was normal and with Mann–Whitney test if distribution was not normal as assessed with the Kolmogorov–Smirnov normality test. Differences are considered statistically significant at *P < 0.05.

Article Snippet: NCL , Nucleolin , Hs01066668_m1.

Techniques: Western Blot, Control, MANN-WHITNEY