Journal: Nature Communications

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1038/s41467-024-49139-7

Figure Lengend Snippet: a Representative LI-COR scanning visualization of poly(A) + RNA crosslinked to an m6A antibody or IgG in neurons, immobilized on a nitrocellulose membrane. RNA is visualized via the ligated infrared adaptor. The portion of the membrane excised and used to generate miCLIP libraries is framed in dashed white lines. For both neurons and NPCs, n = 2 technical replicates from 4 independent iPSC lines (biological replicates). NPC = neural precursor cells; Neur = neurons. b Metagene plot showing m6A site distribution in neurons. The motif with the most significant p value according to the HOMER analysis is displayed. P value was calculated within the HOMER package using a binomial distribution test. c Venn diagram displaying the number of m6A sites in neurons and/or NPCs. d m6A sites detected by miCLIP (top) and RNAseq (sequencing reads, bottom) are displayed on the 3’end of three example genes in NPCs and neurons, using Integrative Genomics Viewer (IGV) software. 3’ untranslated region (3ʹUTR) and last exon are marked by a blue box and separated by an arrow. NPC = neural precursor cells; Neur = neurons. e Cumulative distribution plot of the log2 FC in protein expression between IMP1 knockdown and control neurons as detected by MS, was plotted for corresponding RNA classified by the number of IMP1-m6A peaks detected by iCLIP and miCLIP. For MS, n = 3 independent iPSC lines for each condition (biological replicates); for iCLIP, n = 3 technical replicates from 3 independent iPSC lines (biological replicates); for miCLIP, n = 2 technical replicates from 3 independent iPSC lines + n = 1 technical replicate from 1 independent iPSC line (biological replicates). P values were calculated using two-sided Kolmogorov–Smirnov test comparing 1–2 peaks or 3–5 peaks or 6 and + peaks to 0 peaks. The corresponding P values are reported on the plot. f Number of IMP1 peaks on m6A sites (IMP1-m6A peaks) calculated by superimposing the results of the IMP1 iCLIP and miCLIP analyses in different GO categories determined by the PANTHER analysis. The three microtubule/cytoskeleton/neuronal terms and two cytoskeleton-unrelated terms displayed are determined by the PANTHER analysis shown in Fig. Data presented as boxplots where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed for clarity. Red dots represent the mean. The categories “Cytoskeleton organization”, “Microtubule-based process”, “Neuron projection development” contain many common genes. Black dots represent transcripts which are present in more than one of these categories, while orange dots represent transcripts in a single category. “Positive regulation protein kinase activity” with “Small molecule biosynthetic process” include different transcripts, represented by blue dots. g Barplot showing the percentage of proteins belonging to the “Microtubules-based process” terms as defined by the PANTHER GO classification for each of the following categories: downregulated proteins as defined in Fig. , downregulated proteins where the encoding mRNA is directly bound by IMP1 as defined in Fig. , downregulated proteins where the encoding mRNA is directly bound by IMP1 as defined in Fig. where IMP1 binds to an m6A site as determined by miCLIP. Source data are provided as a Source Data file.

Article Snippet: Rabbit anti-IMP1 (MBL, RN007P), dilution 1:1000 (WB), dilution 1:100 (IF), 5ug for iCLIP rabbit anti-IgG (Proteintech, 30000-0-AP), 5ug for miCLIP and iCLIP rabbit anti-GAPDH (Cell signalling 14C10), 1:1000 (WB), mouse anti-Actb (Sigma, A2228), 1:1000 (WB), rabbit anti-H3 (Abcam, ab201456), 1:2000 (WB), chicken anti-Homer-1 (Synaptic System, 160006), dilution 1:100 (IF), mouse anti-Synaptotagmin1 (Synaptic System, 105011C3), dilution 1:100 (IF), mouse anti-SMI-35 (BioLegend, 835603), dilution 1:100 (IF), rabbit anti-MAP1B (Proteintech, 21633-1-AP), dilution 1:100 (IF), mouse anti-MAP2 (Abcam, ab11267), 1:1000 (WB), chicken anti-MAP2 (Abcam, ab5392), dilution 1:100 (IF), chicken anti-beta III tubulin (Abcam, ab41489), dilution 1:100 (IF), mouse anti-beta IV tubulin (Abcam, ab11315), dilution 1:100 (IF), rabbit anti-DCX (Proteintech, 13925-1-AP), 1:1000 (WB), 1:100 (IF), mouse anti-MAPT (Abcam, ab80579), dilution 1:100 (IF), rabbit anti-CRIPT (Proteintech,11211-1-AP), dilution 1:100 (IF), mouse anti-m6A (Abcam, ab151230), 5ug for miCLIP, rabbit anti-METTL3 (Proteintech, 15073-1-AP), 1:1000 (WB).

Techniques: Membrane, Sequencing, Software, Expressing, Knockdown, Control, Activity Assay

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: PD-L1 is modified by UBL3. ( a ) Co-immunoprecipitation (IP) analysis of using MDA-MB-231 cell lysates expressing Flag-tagged PD-L1 (PD-L1-Flag) and biotinylated-tagged UBL3 (Biotin-UBL3). ( b ) Co-IP was performed using cell lysates expressing PD-L1-Flag or PD-L1 mutants-Flag (C250A, C272A, C250/272A) along with Biotin-UBL3 in MDA-MB-231 cells. IB analysis was conducted using the indicated antibodies. ( c ) the relative intensity of UBL3 modification (Flag/SA-HRP) calculated as the ratio of UBL3-modified PD-L1 signals (> 70 kDa) to input SA-HRP signals, normalized to the mean of cells transfected with Biotin-UBL3 and wild-type PD-L1. Data are presented as mean ± s.e.m., with dots representing individual experiments. One-way ANOVA with Tukey’s multiple comparisons test. ns, not significant, * P < 0.05, ** P < 0.001, *** P < 0.0005, PD-L1 vs. C250A, P = 0.1708; PD-L1 vs. C272A, P = 0.0009; PD-L1 vs. C250/272A, P = 0.0002; C250A vs. C272A, P = 0.0135; C272A vs. C250/272A, P = 0.5847; TMD, transmembrane domain; Flag, Flag-tag; βME-, without 2-mercaptoethanol; βME+, with 2-mercaptoethanol; SA-HRP, streptavidin-horseradish peroxidase.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Modification, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 regulates PD-L1 levels in sEVs. ( a ) Immunoblot (IB) analysis. The cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. ( b ) IB analysis. The cell lysates and sEVs from the stable UBL3 knockdown H1299 cell lines were blotted with various antibodies. CD9 and CD63 were used as sEV markers, as they are tetraspanin proteins abundantly expressed on the sEV surface . Relative intensity of PD-L1 in sEVs was calculated as PD-L1/CD63 and presented as fold change, normalized to the mean value of the respective control (mock for a; shNega for b). Data are presented as mean ± s.e.m., with dots representing individual experiments. Two-tailed unpaired t-test. *** P < 0.005.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Western Blot, Transfection, Knockdown, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: Inhibition of UBL3 modification and PD-L1 sorting by statins. ( a and b ), Effect of pitavastatin treatment on UBL3 modification in WM9 ( a ) and H1299 ( b ) cells. Five hours after transfection, 10 µM pitavastatin was added. ( c and d ), IB analysis of the cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors ( c ) or untransfected ( d ) were blotted with various antibodies. Five hours after gene transfection, 0.2 µM pitavastatin was added. Pit, Pitavastatin. Right panels, the relative intensity of PD-L1 in sEVs (c: PD-L1/CD63 [upper] normalized to the mean of mock − untreated control, and UBL3/CD63 [lower] normalized to the mean of Flag-UBL3 − untreated control; d: PD-L1/CD63 [upper] and UBL3/CD63 [lower], both normalized to the mean of untreated control). Data are presented as mean ± s.e.m., with dots representing individual experiments. As inhibition of UBL3 modification was observed even at low concentrations of pitavastatin (Supplementary Fig. S4b), a low concentration of pitavastatin was used in sEV purification experiments. IB analysis using the indicated antibodies. Flag, Flag-tag; GAPDH, internal control; CD63 and CD9, sEVs markers; PD-L1, endogenous PD-L1; UBL3, endogenous UBL3. βME−, without 2-mercaptoethanol. βME+, with 2-mercaptoethanol. ( c ), one-way ANOVA with Tukey’s multiple comparisons test. ( d ), Two-tailed unpaired t-test. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Inhibition, Modification, Transfection, Control, Concentration Assay, Purification, FLAG-tag, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 and PD-L1 expression levels influence survival in lung cancer patients. ( a ) Kaplan–Meier survival curves for the lung squamous cell carcinoma cohort, stratified by UBL3 and PD-L1 expression. ( b ) Survival curves for the same cohort, stratified by UBL3 and PD-1 expression. Hazard ratios and p-values from log-rank (Mantel–Cox) tests are summarized in Supplementary Figure S7a.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Knockdown of CDKL3 inhibited cellular proliferation and the colony formation ability in TE-1 and KYSE-150 cells. (A) Western blot analysis showed that LV-shCDKL3 significantly downregulated protein levels of CDKL3 compared with LV-shCtrl group after 72 h infection. (B) The Celigo assay and MTT assay confirmed the inhibitory effect of CDKL3 knockdown on the proliferation rate of TE-1 and KYSE-150. (C) The typical Celigo images for the effect of CDKL3-knockdown on the proliferation of TE-1 and KYSE-150 cells. (D) CDKL3 knockdown decreased the colon numbers in TE-1-shCDKL3 and KYSE-150-shCDKL3 cells compared to TE-1-shCtrl and KYSE-150-shCtrl cells, respectively. (E) colonies of ESCC cells stained with crystal violet. Data were represented as the mean ± SD of three separate experiments. *** P < 0.001.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Knockdown, Western Blot, Infection, MTT Assay, Staining

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Knockdown of CDKL3 increased apoptosis in TE-1 and KYSE-150 cells, which was measured by Annexin-V-APC and caspase 3/7 assay. (A) Early apoptotic cells (Annexin-V+) were displayed in the upper and lower right quadrant. Knockdown of CDKL3 increased the early apoptotic rates in TE-1-shCDKL3 and KYSE-150-shCDKL3 cells. (B) Knockdown of CDKL3 remarkably enhanced caspase-3/7 activity in TE-1-shCDKL3 and KYSE-150-shCDKL3 cells. Data were represented as the mean ± SD of three separate experiments, *** P < 0.001.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Knockdown, Activity Assay

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Using transwell-with or without Matrigel assay, CDKL3 knockdown decreased the numbers of migrated and invaded cells in the shCDKL3 group compared with the shCtrl group. Data were represented as the mean ± SD of three separate experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Matrigel Assay, Knockdown

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: CDKL3 knockdown retarded tumor growth in vivo . (A) KYSE-150-shCDKL3 cells generated smaller tumors in nude mice compared to the KYSE-150-shCtrl cells, * P < 0.05. (B) KYSE-150-shCDKL3 cells formed lighter tumors compared to the KYSE-150-shCtrl cells, * P < 0.05. (C) Western blotting analysis of the presence of CDKL3 and ATG5 proteins from the harvested tumors as described for (B) , * P < 0.05. Data were represented as mean ± SD.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Knockdown, In Vivo, Generated, Western Blot

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: ATG5 is a potential target of CDKL3. (A) A scatter plot demonstrated the distribution of the signal intensity between two groups in a Cartesian coordinate plane. The X -axis represents KYSE-150-shCtrl group, and the Y -axis represents the KYSE-150-shCDKL3 group. The dots above the upper parallel line represented the downregulated genes, while the dots under the lower parallel line represented the upregulated genes. (B) Volcano Plot exhibited the significantly differentially expressed genes between the two groups. The X -axis denotes the log2-fold difference and the Y -axis denotes the log10-corrected significant. The red dots represented the differentially expressed genes screened with the absolute value of Fold Change ≥1.5 and FDR < 0.05. (C) The interaction network illustrated the interrelationships among these 22 selected genes surrounding the regulation of CDKL3. The up-regulation gene was red while the down-regulation gene was green. The solid line represented the direct interactions while the broken lines represented the indirect interactions, and an arrow represented the activation. (D) RT-PCR was used to confirm the up-regulated and down-regulated genes induced by CDKL3 knockdown in KYSE-150 cells. (E) The expression patterns of the potential interacting genes in the TCGA database according to tumor histology. (F) Comparison of overall survival of esophageal cancer patients with different genes expression based on TCGA data. Data were represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. controls, respectively.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Knockdown, Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: ATG5 re-expression partly counteracted the anti-tumor effect of CDKL3 knockdown in KYSE-150 cells. (A) After infection, KYSE-150 cells expressed green and red fluorescence, Magnification 100×. (B) Protein level showed that the re-overexpression of ATG5 in KD + OE group and down-expression of ATG5 in KD + NC-2 group (KD + NC). (C) Overexpressed ATG5 partially diminished the anti-tumor effect of CDKL3 knockdown by proliferation assays compared to KD + NC group. (D) The ability of colony formation was restored by ATG5 re-expression compared to KD + NC group. (E) ATG5 re-expression induced caspase-3/7 activity, which was inhibited by CDKL3 knockdown compared to KD + NC group. Data were represented as the mean ± SD of three separate experiments. * P < 0.05; ** P < 0.01, *** P < 0.001. (F) The expression of LC3A/B was analyzed by western blot analysis.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Expressing, Knockdown, Infection, Fluorescence, Over Expression, Activity Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: The expression of CDKL3 and ATG5 is upregulated in ESCC tissues and predicts a poor clinical outcome. (A) typical immunohistological pictures with high expressions of CDKL3 and ATG5 in ESCC. The staining showed predominantly cytoplasmic localization, Magnifications 200×. (B) Representative images of CDKL3 staining according to TNM stage and representative images of ATG5 staining according to the T stage and TNM stage in ESCC patients (magnification, 200×). (C) Kaplan–Meier survival curve was drawn to analyze the prognostic significance of CDKL3 and ATG5 expression in overall survival of ESCC patients.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Differential expression of CDKL3 and ATG5 in 46 paired ESCC and adjacent tissues.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: The relationship between CDKL3 and ATG5 expression status and clinicopathologic features of ESCC.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic factors correlated with OS in ESCC patients.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Association of CDKL3 expression levels with ATG5 expression status.

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma

doi: 10.3389/fonc.2020.01602

Figure Lengend Snippet: Time-dependent ROC curves showed that the combination of CDKL3, ATG5 and TNM stage–based model (named as improved TNM) had better predictive accuracy than TNM stage in ESCC patients (FP, false positive or specificity, TP, true positive or sensitivity).

Article Snippet: Briefly, tissue sections were cut, deparaffinized and rehydrated, and incubated with the rabbit polyclonal anti-CDKL3 antibody and rabbit polyclonal anti-ATG5 antibody (Proteintech, Chicago, IL, United States) at 1/1000 and 1/750 dilutions, respectively.

Techniques:

Journal: The FASEB Journal

Article Title: Deep multilayer brain omics identifies the potential involvement of menopause molecular networks in Gliomas’ disease progression

doi: 10.1096/fj.202200427rr

Figure Lengend Snippet: FIGURE 3 Phosphorylation analysis revealed distinct protein signatures in different groups. (A) Hierarchical clustering analysis of 152 LFQ-phosphorylation intensities (log2) quantified in three repeats of the four groups' proteomes and showed at least p < 0.05 in their abundance between at least two groups. (B) GO enrichment analysis for the category of upregulated and downregulated phosphorylated proteins between sham and OVX groups. (C) GO enrichment analysis for the category of upregulated and downregulated phosphorylated proteins between sham and MENO groups. (D) MAPK signaling pathway: signaling pathways associated with differentiation, proliferation, and inflammation in the menopausal group. (E) Ether lipid metabolism: signaling pathways associated with lipid metabolism in the menopausal group. (F) The mRNA levels of RSK2, JNK, and LPCAT4 were detected in the LN229 cells with estrogen stimulation. Statistical analysis was performed by unpaired T tests, **p < 0.01 compared with the control group. (G) LPCAT4 protein levels were detected in the LN229 cells with estrogen stimulation compared with no-estrogen stimulation. Statistical analysis was performed by unpaired T tests, **p < 0.01 compared with the control group.

Article Snippet: Primary antibodies, rabbit anti JNK (pT183/pY185) (4668, cell signaling), and rabbit anti LPCAT4 (17905- 1- AP, Proteintech) were used to analyze the protein expression levels.

Techniques: Phospho-proteomics, Protein-Protein interactions, Control

Journal: The FASEB Journal

Article Title: Deep multilayer brain omics identifies the potential involvement of menopause molecular networks in Gliomas’ disease progression

doi: 10.1096/fj.202200427rr

Figure Lengend Snippet: FIGURE 5 Identification of upregulated and downregulated proteins in human gliomas. (A) The differentially expressed proteins (top 20) from the postmenopausal brain proteomics database of mice. (B) Expression levels of upregulated and downregulated proteins in human gliomas from TCGA datasets. (C) Overall survival curves for the expression of ACTA2, CAMK2D, FNBP1L, ARL1, HEBP1, CAST, CLIC1, LPCAT4, MAST3, and DOCK9 in Gliomas (Kaplan–Meier Plotter). (D) Correlation analysis of ACTA2, CAMK2D, FNBP1L, ARL1, HEBP1, CAST, CLIC1, LPCAT4, MAST3, and DOCK9 gene expression TMB/MSI. The horizontal axis in the figure represents the expression distribution of the gene, and the ordinate is the expression distribution of the TMB/MSI score. The density curve on the right represents the distribution trend of the TMB/MSI score; the upper-density curve represents the distribution trend of the gene on the top side. The value represents the correlation p-value, correlation coefficient, and correlation calculation method. (E and F) Expression levels of upregulated and downregulated proteins in human gliomas from TCGA datasets by multi-factor analysis including ages and grades.

Article Snippet: Primary antibodies, rabbit anti JNK (pT183/pY185) (4668, cell signaling), and rabbit anti LPCAT4 (17905- 1- AP, Proteintech) were used to analyze the protein expression levels.

Techniques: Expressing, Gene Expression

Journal: The FASEB Journal

Article Title: Deep multilayer brain omics identifies the potential involvement of menopause molecular networks in Gliomas’ disease progression

doi: 10.1096/fj.202200427rr

Figure Lengend Snippet: FIGURE 7 Identification of upregulated and downregulated genes in human postmenopausal brains. (A) The boxplot of the normalized data. Different colors represent different datasets. Rows represent samples, and columns represent the gene expression values in the samples. (B) PCA results after batch removal for multiple datasets. Different colors represent different datasets. As shown in the schematic diagram, five datasets are separated without any intersection. (C) Differential analysis of expressed genes in the brains between postmenopausal mice and postmenopausal women. (D) The expression distribution of ACTA2, CAMK2D, FNBP1L, ARL1, HEBP1, CAST, CLIC1, LPCAT4, MAST3, and DOCK9 in the brains of pre (age: 20–50) and postmenopausal (age > 50) women. **p < 0.01, and ***p < 0.001, asterisks (*) stand for significance levels. The statistical difference between the two groups was compared through the T test.

Article Snippet: Primary antibodies, rabbit anti JNK (pT183/pY185) (4668, cell signaling), and rabbit anti LPCAT4 (17905- 1- AP, Proteintech) were used to analyze the protein expression levels.

Techniques: Gene Expression, Expressing

Journal: The FASEB Journal

Article Title: Deep multilayer brain omics identifies the potential involvement of menopause molecular networks in Gliomas’ disease progression

doi: 10.1096/fj.202200427rr

Figure Lengend Snippet: FIGURE 9 Gene expression study of blood monocytes in pre- and postmenopausal females. (A–D) Upregulated genes (ARL1, DCUN1D1, PPP2CA, SLC24A3) in blood monocytes. (E–H) Downregulated genes (KRT1, LPCAT4, PCSK1N, HEBP1) in blood monocytes. (I) Sensitivity and specificity comparison of different protein expressions in gliomas associated with glioma prognosis by the area under the ROC curve.

Article Snippet: Primary antibodies, rabbit anti JNK (pT183/pY185) (4668, cell signaling), and rabbit anti LPCAT4 (17905- 1- AP, Proteintech) were used to analyze the protein expression levels.

Techniques: Gene Expression, Comparison

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Endogenous Chi3l3 expression in the adult SVZ during EAE. a Chi3l3 gene expression in the subventricular zone (SVZ) of healthy control mice (HC; n = 8), preclinical EAE (6 days post immunization (dpi; n = 6), EAE onset (13dpi; n = 6), acute EAE (14 dpi; n = 6), initial recovery (18 dpi; n = 8) and chronic EAE (36 dpi, n = 6) measured by qRT-PCR (multiple t -tests with Dunn–Bonferroni’s multiple comparison test). b – e Confocal images of the SVZ of representative healthy controls (HC), acute EAE mice (12–14 dpi). Sections were immunostained for the nuclear marker TO-PRO-3 (blue), Chi3l3 (red) and the microglia/macrophage marker CD11b (green, b – e ) and the lymphocyte common antigen CD45 (blue, d ). Chi3l3 expression in the SVZ was only detected during acute EAE and was localized to CD11b-positive cells (yellow arrowheads) within the corpus callosum (CC) c and lesions d , where they co-expressed CD45 at high (yellow arrowhead) and low levels (white arrowhead). Dashed lines mark the wall of the lateral ventricle (LV). Scale bar, 200 μm. e Ortho-view images of representative Chi3l3 + /CD11b + double-positive cells exhibiting round (upper panel) and ramified (lower panel) morphology. (mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Expressing, Quantitative RT-PCR, Marker

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 induces a pro-oligodendrogenic transcriptional and signaling profile. a , b Gene expression of the pro-oligodendroglial transcription factors Olig1 , Olig2, and Sox10 after 36 h a and negative transcriptional regulators Id2, Id4, and Hes5 after 24 h b in differentiating neural stem cells (NSCs) post exposure to Chi3l3 or PBS (control). c Mean fluorescence intensity (MFI) quantification of individual NSCs, activated by Chi3l3 or PBS (control), fixed at 5, 15, and 30 min and immunostaining for phosphorylation (p)- specific antibodies to various signaling proteins. d Ingenuity pathway analysis showing interconnection between Chi3l3-mediated signaling events ( Ptk2b (Pyk2), MAPK14 (p38MAPK), PLCG2 (PLCγ2), RAF1 (c-Raf), ERK1/2, PI3K p85 and transcriptional events ( Olig1, Olig2, Sox10, Hes5 , Id2 , Id4 ). e , f Gene expression of Olig2 in differentiating NSCs in the presence of Pyk2 kinase inhibitor PF-431396 e or NSCs, transfected with control shLVPs or Ptk2b (Pyk2) shLVPs f after 36 h of exposure to Chi3l3 or PBS. g , h Gene expression of Olig2 in differentiating NSCs in presence of MEK-Erk inhibitor U0126 g or NSCs, transfected with LVPs containing GFP (control) or dnErk2-GFP h after 36 h of exposure to Chi3l3 or PBS. Values were normalized to housekeeping genes and expressed as percent change to PBS-treated control NSCs. i Quantification of NG2 + oligodendrocyte precursor cells, derived from NSCs differentiated for 3 days in the presence ( + ) or absence (−) of PF-431396 and Chi3l3 ( + ) or PBS (−). j Quantification of NG2 + oligodendrocyte precursor cells derived from differentiating NSCs, transfected with control shLVPs or Ptk2b shLVPs (Pyk2), and treated with Chi3l3 ( + ) or PBS (−). Pyk2 signaling deficiency significantly reduced Chi3l3-induced oligodendrogenesis. Values are expressed as percent change to the corresponding PBS-treated group. Two-tailed Student’s t test was performed for a – c , one-way ANOVA followed by Turkey’s post hoc test was performed for e – j ; Data are representative of three independent experiments. For n numbers please refer the Methods section; mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Expressing, Fluorescence, Immunostaining, Transfection, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 promotes oligodendrogenesis by inducing EGFR signaling. a ingenuity pathway analysis showing membrane-bound receptors (EGFR, FGFR1, DLG4 (PSD95), NTRK2, FYN) upstream of Chi3l3-induced MAPK signaling (Ptk2b (Pyk2), MAPK14 (p38MAPK), PLCG2 (PLCγ2), RAF1 (c-Raf), ERK1/2, PI3K p85) and pro-oligodendrogenic transcriptional pattern ( Olig1, Olig2, Sox10, Hes5 , Id2 , Id4 ). b Mean fluorescence intensity (MFI) quantification of individual neural stem cells (NSCs), activated by Chi3l3, EGF, or PBS (control) for 15 min, and immunostaining for phospho (p)-specific EGFR (Tyr1068). One-way-ANOVA with Dunn’s multiple comparison test; c Quantification of NSC-derived NG2 + oligodendrocyte precursor cells after 3 days of differentiation in the presence of Chi3l3 ( + ) or PBS (−) and EGFR kinase inhibitor AZD8931 ( + , left) and Gefitinib ( + , right) or DMSO (−). One-way ANOVA with Bonferroni’s multiple comparison test; d Quantification of NG2 + oligodendrocyte precursor cells derived from differentiating NSCs, transfected with control shLVPs or EGFR shLVPs and treated with Chi3l3 ( + ) or PBS (−). Deficiency in EGFR signaling significantly reduced Chi3l3-induced oligodendrogenesis. One-way ANOVA with Bonferroni’s multiple comparison test; e Mean fluorescence intensity (MFI) quantification of NSCs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control) in the presence of EGFR inhibitor AZD8931 for 15 min, and immunostaining for phospho (p)-specific Pyk2 (Tyr402). One-way ANOVA with Bonferroni’s ( d , neuroblasts, red) test; f Mean fluorescence intensity (MFI) quantification of NSCs transfected with control shLVPs or EGFR shLVPs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control for 15 min, and immunostained for phospho (p)-specific Pyk2 (Tyr402). Deficiency in EGFR signaling reduced Pyk2 phosphorylation in neural stem cells. All values are expressed as percent change to PBS-treated NSCs. One-way ANOVA with Bonferroni’s multiple comparison test; mean ± s.e.m; * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Fluorescence, Immunostaining, Derivative Assay, Transfection, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 infusion induces pro-oligodendrogenic fate of subventricular zone neural stem cells in vivo. a Protocol of BrdU labeling via intraperitoneal (i.p.) injection and Chi3l3 administration. b – f Representative confocal images b , d and quantification c , e , f of the ipsilateral (IL) or contralateral (CL) subventricular zone (SVZ) of naive mice infused with Chi3l3 or PBS (vehicle) and immunostained for BrdU (green) and NG2 ( b , oligodendrocyte precursor cells, red) or Dcx ( d , neuroblasts, red). IL SVZ of Chi3l3-treated mice were compared with IL SVZ of vehicle-treated mice as well as the CL site of the same mouse. Dashed lines mark the ventricular wall of the right or left lateral ventricle (RLV; LLV). Scale bar (left), 50 μm. Double-positive cells are indicated by yellow and white arrowheads and representative cells (box) are shown as high magnification images (right). Scale bar (right), 5 μm. Quantifications show a significantly higher percentage of BrdU/NG2 double-positive cells c and reduced percentage of BrdU + /Dcx + cells e in the ipsilateral SVZ of Chi3l3 infused mice indicating pro-oligodendrogenic modulation of SVZ stem cell fate choice in vivo. f Quantification of the total number of BrdU labeled cells/mm 2 in the SVZ showed no significant difference between Chi3l3 or vehicle infusion in the ipsilateral or contralateral SVZ. (One-way ANOVA with Turkey’s post hoc test; c n = 2 mice (Control IL, CL), n = 4 (Chi3l3 IL), and n = 3 (chi3l3 CL); e n = 2 mice per group, n = 4 mice per group; mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: In Vivo, Labeling, Injection

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Subventricular zone-derived Chi3l3 affects EAE severity and oligodendrogenesis. a Protocol of shRNA lentiviral particle (LVP) administration, BrdU injection, and EAE induction. b Clinical score (left) and linear regression curves (right) of mice treated with control shRNA LVPs (black line) or Chi3l3 shRNA LVPs (red line) and immunized with PLP 139–151 . Dashed lines indicate 95% confidence intervals. ( n = 23 control shRNA LVP and 27 Chi3l3 shRNA LVP mice per group; two-way ANOVA; c Quantifications of percentage (left) and number (right) of NG2 + /BrdU + double-positive cells in the ipsilateral (IL) and contralateral (CL) SVZ of Chi3l3 shLVP and control shLVP mice killed at onset of disease in vivo. ( n = 4 per group; two-tailed Student’s t test; d Quantifications of and number of NG2 + cells in the ipsilateral (IL) and contralateral (CL) SVZ of Chi3l3 shLVP and control shLVP mice sacrificed during chronic EAE ( n = 8 control shRNA mice, n = 6 Chi3l3 shRNA mice; two-tailed Student’s t test. e , f Localization (left) and quantification of inflammatory lesions in brain sections (middle) and spinal cord sections (right) of Chi3l3 shLVP and control shLVP mice killed at EAE onset ( e ; n = 4 mice control shRNA, n = 5 mice Chi3l3 shRNA (left, brain), n = 5 mice per group (right, spinal cord; two-tailed Student’s t-test; mean ± s.e.m) and during chronic EAE ( f , n = 7 mice control shRNA mice, n = 8 Chi3l3 shRNA mice (left, brain) and n = 8 mice control shRNA mice, n = 6 Chi3l3 shRNA mice (right, spinal cord); two-tailed Student’s t test). Schematic diagram showing localization of inflammatory lesions of individual mice, each color represents an individual mouse. (mean ± s.e.m.; * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Derivative Assay, shRNA, Injection, In Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Proposed model of Chi3l3-induced oligodendrogenesis. In the central nervous system, Chi3l3 is expressed and secreted by alternatively activated myeloid cells (MC), including resident microglia and infiltrating macrophages. These cells are located in the corpus callosum (CC) and in close proximity to the subventricular zone (SVZ) of the lateral ventricle (LV). Chi3l3 binds to epidermal growth factor receptor (EGFR) on the surface of endogenous neural stem cells (NSCs) and activates MEK/ERK signaling and a pro-oligodendrogenic transcription factor pattern in a Pyk2-dependent manner, leading to oligodendrogenesis

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques:

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Expression of PDCL3 in pan-cancer tissues. A Analysis of PDCL3 expression in various cancers using the TIMER database. B Analysis of the differential protein expression of PDCL3 in tumors and normal tissues using the CPTAC database. C Expression of PDCL3 in different cancers and its association with prognosis. D Kaplan–Meier survival curves for patients with different types of cancer. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 (Student’s t test)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Baseline characteristics of liver cancer patients (analysis of variance)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Overexpression of PDCL3 in LIHC. A Expression levels of PDCL3 in LIHC tumor tissues and normal tissues. B Expression levels of PDCL3 in LIHC (hepatocellular carcinoma) and matched adjacent normal tissues. C – E Relationship between PDCL3 overexpression and clinical, pathological, and histological staging. F Correlation of PDCL3 with overall survival rate. G PDCL3 staining from IHC results provided by the HPA database. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 (Student’s t test)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Over Expression, Expressing, Staining

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Prognostic relevance of PDCL3 in LIHC. A – C Relationship between PDCL3 expression and patient prognosis in terms of OS (overall survival), DSS (disease-specific survival), and PFS (progression-free survival). D ROC curves for PDCL3 and LIHC prognosis. E – G Time-dependent ROC curves for PDCL3 and LIHC patient prognosis. H Clinical prognostic risk score for PDCL3. I Calibration plot for model accuracy assessment

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Functional enrichment analysis of PDCL3 in LIHC. A Volcano plot of molecules related to PDCL3 expression in LIHC. B – D Analysis results of BP (biological process), CC (cellular component), and MF (molecular function) related to molecules associated with PDCL3 in LIHC. E Pathway analysis related to PDCL3 in LIHC. F Hill plot of pathway enrichment analysis related to PDCL3

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Functional Assay, Expressing

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Relationship between PDCL3 expression and immune infiltration in LIHC. A Relationship between PDCL3 expression and macrophage infiltration. B Correlation of PDCL3 expression with the expression of various immune checkpoint genes in LIHC. C Expression levels of individual immune checkpoint genes in different PDCL3 expression groups. D Infiltration of immune cells in different PDCL3 expression groups. E Analysis of the correlation between high PDCL3 expression, immune infiltration, and adverse prognosis in LIHC patients. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 (analysis of variance)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: Differential expression of PDCL3 in adjacent non-cancerous tissues and liver cancer tissues. A Hematoxylin and eosin (H&E) staining images of normal tissues and liver cancer tissues. B Immunofluorescence images depicting the distribution of PDCL3 in normal tissues and liver cancer tissues. C Immunohistochemistry staining images showing PDCL3 in normal tissues and liver cancer tissues. D H-scores and IRS scores of PDCL3 protein IHC in normal tissues and liver cancer tissues. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 (Student’s t test)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Quantitative Proteomics, Staining, Immunofluorescence, Immunohistochemistry

Journal: European Journal of Medical Research

Article Title: PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

doi: 10.1186/s40001-024-01787-7

Figure Lengend Snippet: PDCL3 promotes proliferation, migration, invasion, and colony formation capacity of LIHC cells in vitro. A Expression levels of PDCL3 in different cell lines. B Interference efficiency of PDCL3 detected using the protein imprinting method. C Cell proliferation rate determined by the CCK-8 method. D , E Results and analysis of wound healing and transwell experiments. F Results and analysis of colony formation experiment.* p value < 0.05; ** p value < 0.01; *** p value < 0.001 (Student’s t test)

Article Snippet: Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)

Techniques: Migration, In Vitro, Expressing, CCK-8 Assay

Journal: Scandinavian journal of immunology

Article Title: Stimulation of naïve monocytes and PBMCs with coagulation proteases results in thrombin-mediated and PAR-1-dependent cytokine release and cell proliferation in PBMCs only.

doi: 10.1111/sji.12033

Figure Lengend Snippet: Figure 1: Expression of mRNA’s encoding PAR-1, PAR-2, PAR-3, and PAR-4 in freshly isolated naïve CD14+ monocytes.

Article Snippet: FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing, Isolation, Polymerase Chain Reaction

Journal: Scandinavian journal of immunology

Article Title: Stimulation of naïve monocytes and PBMCs with coagulation proteases results in thrombin-mediated and PAR-1-dependent cytokine release and cell proliferation in PBMCs only.

doi: 10.1111/sji.12033

Figure Lengend Snippet: Figure 2: Freshly isolated naïve monocytes express PAR-1, PAR-3, and PAR-4, but do not express PAR-2 or TF.

Article Snippet: FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation

Journal: Scandinavian journal of immunology

Article Title: Stimulation of naïve monocytes and PBMCs with coagulation proteases results in thrombin-mediated and PAR-1-dependent cytokine release and cell proliferation in PBMCs only.

doi: 10.1111/sji.12033

Figure Lengend Snippet: Figure 3: PAR-1, PAR-3, and PAR-4 expression on naïve monocytes is not altered by stimulation with FVIIa, TF+rFVIIa, TF+rFVIIa+FX, FX, FXa, Thrombin, or LPS.

Article Snippet: FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing

Journal: Scandinavian journal of immunology

Article Title: Stimulation of naïve monocytes and PBMCs with coagulation proteases results in thrombin-mediated and PAR-1-dependent cytokine release and cell proliferation in PBMCs only.

doi: 10.1111/sji.12033

Figure Lengend Snippet: Figure 4: Mean fluorescence of PAR-1, PAR-3, and PAR-4 on naïve monocytes is not altered by FVIIa, TF+rFVIIa, TF+rFVIIa+FX, FX, FXa, Thrombin, or LPS.

Article Snippet: FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Fluorescence