Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Theranostics

Article Title: Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway

doi: 10.7150/thno.71585

Figure Lengend Snippet: Specific endothelial S1pr2 loss-of-function enhances post-ischemic blood flow perfusion and angiogenesis in hindlimbs. A , Representative laser Doppler images show improved blood flow perfusion in S1pr2 ECKO mice compared with WT control mice. B , Cumulative results for WT mice (n = 8) and S1pr2 ECKO mice (n = 8) are shown as the ratio of blood flow in the ischemic limb to that in the non-ischemic limb at each time point. C-D , Representative images of isolectin-B4 staining of gastrocnemius muscles in WT and S1pr2 ECKO mice after sham or HLI operation (C) , with quantification of capillary density in gastrocnemius muscle after sham or HLI operation (D) (n = 6). E-F , Representative images of α-SMA staining of gastrocnemius muscles WT and S1pr2 ECKO mice after sham or HLI operation (E) , with quantification of arteriole density in gastrocnemius muscle (F) (n = 6). G-H , Representative images of H&E staining of gastrocnemius muscles in WT and S1pr2 ECK O mice (G) , with quantification of muscle fiber area after sham or HLI operation (H) (n = 5). I-J, Representative images of Sirius red-stained of gastrocnemius muscles in WT and S1pr2 ECKO mice (I), with quantification of the percentage of fibrotic tissue in gastrocnemius muscles (J) (n = 5). K-M, Functional assessment of ischemic muscle over follow-up. Cumulative results for WT mice and S1pr2 ECKO mice are shown graphically as Tarlov score (K), ischemia score (L), and ambulatory impairment score (M) (n = 8). Scale Bars: C and E, 50 μm; G and I, 100 μm. Data are mean ± SEM. n.s indicates not significant. *P < 0.05; **P < 0.01.

Article Snippet: Immunostaining was performed on cryostat 8 μm-thick sections by using various antibodies, including anti-smooth muscle actin antibody (Proteintech, #00098013), biotinylated-isolectin B4 antibody (IB4, Vector Laboratories, B-1205), and their corresponding secondary antibodies.

Techniques: Staining, Functional Assay

Journal: Theranostics

Article Title: Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway

doi: 10.7150/thno.71585

Figure Lengend Snippet: Specific endothelial S1pr2 gain-of-function reduces post-ischemic angiogenesis and blood flow perfusion after HLI. A, Generation of EC-specific S1pr2 gain-of-function mice. B, Relative mRNA expression levels of S1pr2 in ECs of WT and S1pr2 ECTg mice (n = 3). C-D, Western blotting analysis of S1pr2 protein levels in ECs from S1pr2 ECTg mice or WT control mice, with their quantification (n = 3). E, Representative laser Doppler images show impaired blood perfusion in S1pr2 ECTg mice compared with WT control mice. F, Cumulative results for WT mice (n = 8) and S1pr2 ECTg mice (n = 8) are shown as the ratio of blood flow in the ischemic limb to that in the non-ischemic limb at each time point. G-H, Representative images of isolectin-B4 staining of gastrocnemius muscles in WT and S1pr2 ECTg mice ( G ), with quantification of capillary density in gastrocnemius muscles after sham or HLI operation ( H ) (n = 6). I-J , Representative images of α-SMA staining of gastrocnemius muscles in WT and S1pr2 ECTg mice ( I ), with quantification of arteriole density in limb muscle after sham or HLI operation ( J ) (n = 6). K-L, Representative images of H&E staining of gastrocnemius muscles in WT and S1pr2 ECTg mice ( K ), with quantification of muscle fiber area after sham or HLI operation ( L ) (n = 5). M-N , Representative images of Sirius red-stained of gastrocnemius muscles in WT and S1pr2 ECTg mice (M), with quantification of the percentage of fibrotic tissue in muscle ( N ) (n = 5). O-Q, Functional assessment of ischemic muscle over follow-up. Cumulative results for WT mice and S1pr2 ECTg mice are shown graphically as Tarlov score ( O ), ischemia score ( P ), and ambulatory impairment score ( Q ) (n = 8). Scale Bars: G and I , 50 μm; K and M , 100 μm. Data are mean ± SEM. n.s indicates not significant. *P < 0.05; **P < 0.01.

Article Snippet: Immunostaining was performed on cryostat 8 μm-thick sections by using various antibodies, including anti-smooth muscle actin antibody (Proteintech, #00098013), biotinylated-isolectin B4 antibody (IB4, Vector Laboratories, B-1205), and their corresponding secondary antibodies.

Techniques: Expressing, Western Blot, Staining, Functional Assay

Journal: Theranostics

Article Title: Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway

doi: 10.7150/thno.71585

Figure Lengend Snippet: Pharmacological inhibition of S1pr2 by JTE013 improves post-ischemic angiogenesis and enhances blood flow perfusion in hindlimbs. A , Representative laser Doppler images show improved blood flow perfusion in JTE013-treated mice compared with control mice. B, Cumulative results for control mice (n = 6) and JTE-treated mice (n = 6) are shown graphically as the ratio of blood flow in the ischemic limb to that in the non-ischemic limb at each time point. C-D, Representative images of isolectin-B4 staining of gastrocnemius muscles in control mice and JTE013-treated mice, with quantification of capillary density in gastrocnemius muscles after sham or HLI operation (n = 5). E-F, Representative images of α-SMA staining of gastrocnemius muscles in control mice and JTE013-treated mice (E) , with quantification of arteriole density in gastrocnemius muscles after sham or HLI operation (F) (n = 5). G-H, Representative images of H&E staining of gastrocnemius muscles in control mice and JTE013-treated mice (G) , with quantification of muscle fiber area after sham or HLI operation (H) (n = 5). I-J, Representative images of Sirius red-stained of gastrocnemius muscles in control mice and JTE013-treated mice (I) , with quantification of the percentage of fibrotic tissue in muscle (J) (n = 5). K-M, Functional assessment of ischemic muscle over follow-up. Cumulative results for control mice and JTE013-treated mice are shown graphically as Tarlov score (K) , ischemia score (L) , and ambulatory impairment score (M) (n = 6). N-P, Western blotting of AKT or eNOS activation status in hindlimbs of mice treated with JTE013 or DMSO and quantification in the indicated groups (n = 4). Scale Bars: C and E , 50 μm; G and I ,100 μm. Data are mean ± SEM. n.s indicates not significant. *P < 0.05; **P < 0.01.

Article Snippet: Immunostaining was performed on cryostat 8 μm-thick sections by using various antibodies, including anti-smooth muscle actin antibody (Proteintech, #00098013), biotinylated-isolectin B4 antibody (IB4, Vector Laboratories, B-1205), and their corresponding secondary antibodies.

Techniques: Inhibition, Staining, Functional Assay, Western Blot, Activation Assay

Journal: Theranostics

Article Title: Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway

doi: 10.7150/thno.71585

Figure Lengend Snippet: RGD-peptide magnetic nanoparticle EC target delivery of S1pr2-siRNA enhances post-ischemic angiogenesis and blood flow perfusion in the hindlimb. A, Perlus staining shown that RGD-Fe 3 O 4 magnetic nanoparticles (arrowhead) are successfully delivered to hindlimb ECs (n = 3). B-C, RGD-nanoparticle packaging S1pr2-siRNA target delivery reduces the expression of S1pr2 (B), as shown by RT-qPCR in hindlimb ECs, with no significant change of S1pr2 expression in the remaining hindlimb components when ECs were removed (C) (n = 3). D, Representative laser Doppler images show improved blood flow perfusion in mice treated with S1pr2-siRNA compared with those treated with scramble-siRNA. E, Cumulative results for mice treated with S1pr2-siRNA (n = 6) or scramble-siRNA (n = 6) are shown graphically as the ratio of blood flow in ischemic limb to that in the non-ischemic limb at each time point. F-G, Representative images of isolectin-B4 staining of gastrocnemius muscles in mice treated with S1pr2-siRNA or scramble-siRNA (F) , with quantification of capillary density in gastrocnemius muscles after sham or HLI operation (G) (n = 5). H-I, Representative images of α-SMA staining of gastrocnemius muscles in mice treated with S1pr2-siRNA or scramble-siRNA (H) , with quantification of arteriole density in gastrocnemius muscles after sham or HLI operation (I) (n = 5). J-K, Representative images of H&E staining of gastrocnemius muscles in mice treated with S1pr2-siRNA or scramble-siRNA (J) , with quantification of muscle fiber area after sham or HLI operation (K) (n = 5). L-M, Representative images of Sirius red-stained of gastrocnemius muscles in mice treated with S1pr2-siRNA or scramble-siRNA (L) , with quantification of the percentage of fibrotic tissue in muscle (M) (n = 5). N-P, Functional assessment of ischemic muscle over follow-up. Cumulative results for mice treated with S1pr2-siRNA or scramble-siRNA are shown graphically as Tarlov score (N) , ischemia score (O) , and ambulatory impairment score (P) (n = 6). Scale Bars: F and H, 50 μm; J and L,100 μm. Data are mean ± SEM. n.s indicates not significant. *P < 0.05; **P < 0.01.

Article Snippet: Immunostaining was performed on cryostat 8 μm-thick sections by using various antibodies, including anti-smooth muscle actin antibody (Proteintech, #00098013), biotinylated-isolectin B4 antibody (IB4, Vector Laboratories, B-1205), and their corresponding secondary antibodies.

Techniques: Staining, Expressing, Quantitative RT-PCR, Functional Assay

Journal: Cell reports

Article Title: ΔN-Tp63 Mediates Wnt/β-Catenin-Induced Inhibition of Differentiation in Basal Stem Cells of Mucociliary Epithelia

doi: 10.1016/j.celrep.2019.08.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mouse anti-Muc5B (1:500, HS) , Santa Cruz Biotech , sc-393952.

Techniques: Recombinant, RNA Library Preparation, cDNA Synthesis, SYBR Green Assay, Quantitative RT-PCR, Software

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: Lactucopicrin (LCP) inhibits OA progression and cartilage degeneration in mice . ( A ) Representative images of mouse articular cartilage stained with H&E, Safranin O/Fast Green; CC: calcified cartilage, and HC: hyaline cartilage. ( B ) Quantification of OA severity using the OARSI histological scoring system . ( C–E ) qRT-PCR analysis of core OA molecular markers: Col10a1/ColX (collagen, type x, alpha 1), Mmp13 , and Adamts5 . ( F ) WB analysis of OA markers: COL2A1 (collagen type ii, alpha 1) and ADAMTS5. ( G–I ) qRT-PCR detection of senescence-associated genes: Cdkn1a/p21, Cdkn2a/p16 INK4a , and Gadd45 . ( J ) WB analysis of senescence-related proteins: CDKN2A/p16 INK4a , CDKN1A/p21, TRP53/p53, and γH2AX. ( K-L ) Immunohistochemical staining of γH2AX in cartilage tissue and quantification of γH2AX-positive cells (%). Scale bar: 100 µm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lactucopicrin , MedChemExpress , 65,725–11-3.

Techniques: Staining, Quantitative RT-PCR, Immunohistochemical staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Ndufc2 is differentially modulated in brains of SHRSP under stroke‐permissive diet. Evidence of complex I dysfunction in brains of SHRSP . A, Heatmap represents expression differences between samples in a color scale ranging from red (up‐regulated) to blue (down‐regulated). Probe sets representing Ndufc2 are marked. Two‐way ANOVA analysis revealed significant Ndufc2 effects in strain ( P =6.96E −05 ), diet ( P =8.29E −10 ), and interaction ( P =2.79E −03 ; n=5 for each strain at each diet). Corresponding densitometric analysis for Ndufc2 expression levels in brains of SHRSP and SHRSR under either RD or JD is shown in (B). C, Confirmation of Ndufc2 differential expression by RT‐PCR in the same tissue samples; * P <0.0001 for each ratio indicated in the figure (n=6 for each group). D, Representative western blot of Ndufc2 expression levels, confirming reduced expression in brains of SHRSP under JD . E, Representative western blot of BNG (n=3) and corresponding densitometric analysis (F). Δ P <0.01 for SHRSP JD versus both SHRSP RD and SHRSR JD . G, Complex I activity measurement; (H) mitochondrial membrane potential assessment. * P <0.0001 for SHRSP JD versus all other samples (I). ATP‐ level measurement in the same tissue samples. Oligomycin was used to confirm functionality of ATP assay as an inhibitor of oxidative phosphorylation. Valinomycin was used as a mitochondrial ATP ase inhibitor. * P <0.0001 for SHRSP JD versus all other samples and for each inhibited experimental point versus the noninhibited corresponding sample (n=6 for each group). Values are expressed as means± SD . Ctrl indicates control; Hsp60, heat shock protein 60; JD, Japanese‐style stroke‐permissive diet; RD, regular diet; RT‐PCR, reverse‐transcriptase polymerase chain reaction; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR .

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Expressing, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Membrane, ATP Assay, Phospho-proteomics, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: In vitro Ndufc2 silencing in A10 cells. A, Confirmation of Ndufc2 silencing by RT‐PCR and western blot; * P <0.0001 for each si RNA versus baseline (n=6). B, Representative western blot of BNG (n=3) in silenced versus nonsilenced A10 cells. All complexes belonging to OXPHOS are identified on the right side; Hsp60 was used to normalize protein expression level. C, Densitometric analysis of BNG . * P <0.0001 for each si RNA versus both baseline and negative CTRL . D, Assessment of complex I activity. E, Mitochondrial membrane potential assessment. * P <0.0001 for each si RNA versus both baseline and negative CTRL (n=6). F, Measurement of ATP levels in silenced versus nonsilenced cells and both in the absence and in the presence of oligomycin and valinomycin (to confirm functionality of the ATP assay as specified above). * P <0.0001 for each si RNA versus baseline and versus negative CTRL , and for each inhibited point versus the noninhibited corresponding sample (n=6 for each group). G, Assessment of resctive oxygen species levels; * P <0.0001 for each si RNA versus both baseline and negative CTRL (n=6 for each group). H, Representative western blot (n=3) of Nf‐κ B p65 and of its inhibitor, IκB, in silenced versus nonsilenced cells, showing that increases of Nf‐κ B p65 expression are accompanied by decreases of IκB expression levels. I, FACS analysis and corresponding percent values of necrotic cells (J). * P <0.0001 for each si RNA versus both baseline and negative CTRL (n=5). Values are expressed as means±SD. Ctrl indicates control; DCHF, 2′,7′‐dichlorodihydrofluorescein; FACS, fluorescence‐activated cell sorting; Hsp60, heat shock protein 60; IκB, nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor; Nf‐κB, nuclear factor kappa B; RT‐PCR, reverse‐transcriptase polymerase chain reaction.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Activity Assay, Membrane, ATP Assay, Control, Fluorescence, FACS, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Expression levels of proteins involved in necrosis in Ndufc2‐silenced as compared to nonsilenced A10 cells. A, MAPK p38; (B) JNK 1; and (C) c‐Jun. Number of western blots for each protein=3. Densitometric analyses are reported on the right side. * P <0.0001 and # P <0.001 for each si RNA versus baseline and negative CTRL . Ctrl indicates control; JNK1, c‐Jun N‐terminal kinase 1; MAPK, mitogen‐activated protein kinase.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Expressing, Western Blot, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Molecular analyses of brains of all strains at 6 weeks of age. A through C, Brains of heterozygous SHR ‐ Ndufc2 em2Mcwi showed significantly lower amount of Ndufc2 mRNA and protein expression, as compared to both parental SHRSR and SHR ‐ Ndufc2 em1Mcwi . Δ P <0.01 versus SHRSR ; * P <0.0001 versus all other samples (n=6 for each group). D through G, Representative western blot of BNG (n=3) showing defective complex I assembly only in heterozygous SHR ‐ Ndufc2 em2Mcwi rats. Corresponding densitometric analyses are shown on the right side of each blot. * P <0.0001 versus all other samples. H, Complex I activity and (I) ATP levels were comparable among all strains at this age (n=3). Values are expressed as means±SD. Ctrl indicates control; het, heterozygous; Hsp60, heat shock protein 60; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Expressing, Western Blot, Activity Assay, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Molecular analyses in brains of both wild‐type and heterozygous SHR ‐Ndufc2 em2Mcwi lines at the end of 4 weeks exposure to either RD or JD . A, Brain Ndufc2 expression in both wild‐type and heterozygous SHR ‐Ndufc2 em2Mcwi is compared to that of RD ‐fed parental SHRSR ; * P <0.0001 for each indicated ratio (n=5). B, Ndufc2 protein expression levels in brains of wild‐type and heterozygous SHR ‐ Ndufc2 em2Mcwi (number of western blots=3) with corresponding densitometric analysis (C); * P <0.0001 for heterozygous SHR ‐ Ndufc2 em2Mcwi upon RD and JD versus wild type. D and E, Representative western blots of BNG in wild‐type and heterozygous SHR ‐ Ndufc2 em2Mcwi (n=3 each) with corresponding densitometric analysis. * P <0.0001 for heterozygous SHR ‐ Ndufc2 em2Mcwi upon RD and JD versus wild type. F, Complex I activity measurement; (G) mitochondrial membrane potential assessment; * P <0.0001 for heterozygous SHR ‐ Ndufc2 em2Mcwi upon JD versus RD and versus wild‐type line at both RD and JD (n=5 each). H, ATP levels measurement in both wild‐type and heterozygous rat lines upon RD or JD (n=5 each). * P <0.0001 for heterozygous SHR ‐ Ndufc2 em2Mcwi versus wild‐type line and for each inhibited point versus the not inhibited corresponding sample. Values are expressed as means± SD . BNG indicates Blue Native–PAGE; Ctrl, control; het, heterozygous; Hsp60, heat shock protein 60; JD, Japanese‐style stroke‐permissive diet; RD, geular diet; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Expressing, Western Blot, Activity Assay, Membrane, Blue Native PAGE, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Detection of oxidative stress in brains of SHR ‐Ndufc2 em2Mcwi under either RD or JD . A, Detection of carbonylated proteins in brains of heterozygous SHR ‐ Ndufc2 em2Mcwi at the end of 4 weeks of either RD or JD feeding with corresponding densitometric analysis (graph below). * P <0.0001 for SHRSP versus RD and for heterozygous SHR ‐ Ndufc2 em2Mcwi JD versus RD , as well as versus SHRSP JD . B, Detection of carbonylated proteins in brains of wild‐type SHR ‐ Ndufc2 em2Mcwi at the end of 4 weeks of either RD or JD feeding with corresponding densitometric analysis (graph below); * P <0.0001 for SHRSP JD versus RD and for both versus all other strains (total band). Het indicates heterozygous; JD, Japanese‐style stroke‐permissive diet; RD, geular diet; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Molecular analyses in brains of wild‐type and heterozygous SHR ‐Ndufc2 em2Mcwi at the end of either RD or JD exposure. A through D, Representative western blots of Nf‐ kB p65 and of SOD 2 (n=3) with corresponding densitometric analysis in brains of heterozygous SHR ‐ Ndufc2 em2Mcwi , as compared to parental, lines at the end of 4 weeks of either RD or JD feeding. E through H, Same analyses (n=3) were performed in brains of wild‐type SHR ‐ Ndufc2 em2Mcwi . Densitometric values are expressed as means± SD . Significance values: (B) Δ P <0.01 for SHRSP JD and heterozygous SHR ‐ Ndufc2 em2Mcwi JD versus SHRSR JD . C, * P <0.0001 for heterozygous SHR ‐ Ndufc2 em2Mcwi JD versus SHRSR JD ; for SHRSP JD versus SHRSP RD and versus all other samples; ο P <0.05 for SHRSP JD versus heterozygous SHR ‐ Ndufc2 em2Mcwi JD . F, * P <0.0001 for SHRSP JD versus all other samples; Δ P <0.01 for SHRSP JD versus SHRSP RD . H, # P <0.001 and * P <0.0001 for SHRSP JD versus all other samples. Het indicates heterozygous; JD, Japanese‐style stroke‐permissive diet; Nf‐κB, nuclear factor kappa B; RD, geular diet; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; Sod2, superoxide dismutase 2, mitochondrial; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Molecular analyses in the brains of wild‐type and heterozygous SHR ‐Ndufc2 em1Mcwi at the end of either RD or JD exposure. A, Ndufc2 expression levels as assessed by RT‐PCR . * P <0.0001 for the indicated ratio (n=4 each). B, Representative western blot of Ndufc2 in the 2 lines upon either RD or JD with corresponding densitometric analysis (C), number of western blots=3 for each line. D, Representative western blots of BNG (n=3) with corresponding densitometric analysis (E). (F) Complex I activity assessment (n=3 for each group); (G) ATP levels measurement (n=3 for each group). Densitometric values are expressed as means± SD . Ctrl indicates control; het, heterozygous; Hsp60, heat shock protein 60; JD, Japanese‐style stroke‐permissive diet; RD, geular diet; RT‐PCR, reverse‐transcriptase polymerase chain reaction; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Detection of oxidative stress in the brains of wild‐type and heterozygous SHR ‐Ndufc2 em1Mcwi at the end of either RD or JD exposure. A, Detection of carbonylated proteins using the Oxyblot Protein Oxidation Detection kit (n=3; Millipore, Milan, Italy) in brains of heterozygous SHR ‐ Ndufc2 em1Mcwi versus parental lines at the end of 4 weeks of exposure to either RD or JD . B, Corresponding densitometric analysis with values shown as means± SD . * P <0.0001 for SHRSP JD versus SHRSP RD and for both versus all other samples. Densitometric values are expressed as means± SD . C, Detection of carbonylated proteins using the Oxyblot Protein Oxidation Detection kit (n=3) in brains of wild‐type SHR ‐ Ndufc2 em1Mcwi versus parental lines at the end of 4 weeks of exposure to either RD or JD . D, Corresponding densitometric analysis with values shown as means± SD . * P <0.0001 for SHRSP JD versus SHRSP RD and for both versus all other samples. Het indicates heterozygous; JD, Japanese‐style stroke‐permissive diet; RD, geular diet; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR; wt, wild type.

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Ndufc2‐related stroke phenotype in both animal models and in humans. A, Stroke occurrence in heterozygous SHR ‐ Ndufc2 em2Mcwi line as compared to SHRSP and to all other lines ( SHRSR , wild‐type SHR ‐ Ndufc2 em2Mcwi , heterozygous, and wild‐type SHR ‐ Ndufc2 em1Mcwi ). Comparison of heterozygous SHR ‐ Ndufc2 em2Mcwi versus SHRSR and all SHRSR ‐derived lines was significantly different ( P <0.002). Comparison of heterozygous SHR ‐ Ndufc2 em2Mcwi versus SHRSP was significantly different ( P <0.001). For numbers of animals included in these studies, see Table . Symbols: open triangles= SHRSP ; closed circles=heterozygous SHR ‐ Ndufc2 em2Mcwi ; straight line includes: SHRSR ; wild‐type and heterozygous SHR ‐ Ndufc2 em1Mcwi , wild‐type SHR ‐ Ndufc2 em2Mcwi . B, NDUFC 2 expression assessed by RT‐PCR in peripheral blood lymphocytes in a cohort of young healthy subjects carrying wild‐type (n=6), heterozygous (n=12), and double‐mutant (n=6) genotypes for NDUFC 2/rs11237379. * P <0.0001 for both CT and TT versus CC wild‐type genotype ( t test analysis). Values in the figure are expressed as means± SD . C, Plot showing the logarithmic OR s for stroke in A allele carriers at rs641836, TT genotype carriers at rs11237379, and A/rs641836+ TT /rs11237379 carriers with respect to the other genotypes of NDUFC 2 tag SNP s (at multivariable logistic regression analysis). OD indicates odds ratio; RT‐PCR, reverse‐transcriptase polymerase chain reaction; SHRSP, stroke‐prone spontaneously hypertensive rat; SHRSR, stroke‐resistant SHR .

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Comparison, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: Modulation of Inflammatory Genes in Ndufc2 ‐Silenced A10 Cells

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques: Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

doi: 10.1161/JAHA.115.002701

Figure Lengend Snippet: SBP, BW, and Proteinuria Levels in Parental and Genetically Manipulated Rat Lines Upon JD Feeding

Article Snippet: We studied 3 tagSNPs in NDUFC2: rs641836, rs584981, and rs11237379 by 7900HT real‐time PCR and TaqMan technology assays (c__26529993_10, c___614632_10 and c__2999825_10, respectively; Life Technologies).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Vertical sleeve gastrectomy associates with airway hyperresponsiveness in a murine model of allergic airway disease and obesity

doi: 10.3389/fendo.2023.1092277

Figure Lengend Snippet: mRNA expression in lung tissue. Lung mRNA expression as measured by quantitative RT-PCR of (A) Il13ra2 , (B) Glp1r , (C) Tgfb1 , (D) Muc5ac , (E) Muc5b , n=6-11 mice per group. Grey bars and ▲ = saline-challenged mice; pink bars and ▼ = HDM-challenged mice. (A–E) were analyzed using a Two-way ANOVA with a Tukey and Sidak post-hoc test. *p<0.05, **p<0.01.

Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) was then performed using Applied Biosystems TaqMan Gene Expression Master Mix and TaqMan primers (glyceraldehyde-3 phosphate dehydrogenase [ Gapdh ] Mm99999915_g1, elastin [ Eln ] Mm00514670_m1, collagen type 1 alpha 1 chain [ Col1a1 ] Mm00801666_g1, collagen type 1 alpha 2 chain [ Col1a2 ] Mm00483888_m1, glucagon-like peptide-1 receptor [ Glp1r ] Mm00445292_m1, interleukin-13 receptor alpha 1 [ Il13ra1 ] Mm01302068_m1, interleukin-13 receptor alpha 2 [ Il13ra2 ] Mm00515166_m1, interleukin-4 receptor alpha [ Il4ra ] Mm01275139_m1, transforming growth factor beta 1 [ Tgfb1 ] Mm01178820_m1, mucin 5AC [ Muc5ac ] Mm01276718_m1, and mucin 5B [ Muc5b ] Mm00466391_m1).

Techniques: Expressing, Quantitative RT-PCR, Saline

Journal: Journal of orthopaedic surgery and research

Article Title: Paeoniflorin shows chondroprotective effects under IL-1β stress by regulating circ-PREX1/miR-140-3p/WNT5B axis.

doi: 10.1186/s13018-023-04238-x

Figure Lengend Snippet: Fig. 6 The identification of target of miR-140-3p. A The diagram showed the putative binding sites among miR-140, the wild type of WNT5B 3′UTR (WT-WNT5B 3′UTR) and its mutant (MUT-WNT5B 3′UTR). B Dual-luciferase reporter assay measured luciferase activity of report vector carrying WT-WNT5B 3′UTR or MUT-WNT5B 3′UTR in C28/I2 cells transfected with miR-140-3p or miR-NC. C RIP determined the enrichment levels of WNT5B and miR-140-3p in C28/I2 cells. D–F RT-qPCR and western blotting measured WNT5B mRNA level and protein level in (D, E) tissues in KOA group (n = 30) and Control group (n = 30), and F C28/I2 cells treated with IL-1β (0, 5, 10 and 20 ng/mL) for 24 h. G Pearson’s correlation (r) analysis analyzed the correlation betweenWNT5B mRNA and miR-140-3p in KOA cartilages (n = 30). H, I RT-qPCR and western blotting detected miR-140-3p and WNT5B levels in IL-1β-induced C28/I2 cells pre-transfected with anti-miR-NC, anti-miR-140-3p, miR-NC, or miR-140-3p. *P < 0.05

Article Snippet: The primary antibodies targeting Bcl-2 (sc-7382), Bax (sc-7480), WNT5B (sc-376249), and GAPDH (sc-32233) were purchased from Santa Cruz (Shanghai, China), as well as the secondary antibody targeting mouse IgG (sc-516102).

Techniques: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of orthopaedic surgery and research

Article Title: Paeoniflorin shows chondroprotective effects under IL-1β stress by regulating circ-PREX1/miR-140-3p/WNT5B axis.

doi: 10.1186/s13018-023-04238-x

Figure Lengend Snippet: Fig. 7 Effect of WNT5B on the protective role of paeoniflorin in IL-1β-induced human chondrocytes in vitro. IL-1β-induced C28/I2 cells were pre-transfected with pcDNA-WNT5B (WNT5B) or pcDNA prior to paeoniflorin (50 μM) treatment for 4 h. A Western blotting detected WNT5B protein expression level. B MTT assay assessed cell viability. C EdU assay was performed to analyze cell proliferation. D FCM examined apoptosis rate. E Western blotting measured protein levels of Bcl-2 and Bax. F ELISA determined products of TNF-α and IL-10. *P < 0.05

Article Snippet: The primary antibodies targeting Bcl-2 (sc-7382), Bax (sc-7480), WNT5B (sc-376249), and GAPDH (sc-32233) were purchased from Santa Cruz (Shanghai, China), as well as the secondary antibody targeting mouse IgG (sc-516102).

Techniques: In Vitro, Transfection, Western Blot, Expressing, MTT Assay, EdU Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of orthopaedic surgery and research

Article Title: Paeoniflorin shows chondroprotective effects under IL-1β stress by regulating circ-PREX1/miR-140-3p/WNT5B axis.

doi: 10.1186/s13018-023-04238-x

Figure Lengend Snippet: Fig. 8 Paeoniflorin modulated circ-PREX1/miR-140-3p/WNT5B axis in IL-1β-induced human chondrocytes in vitro. A Western blotting examined WNT5B protein level in IL-1β-induced C28/I2 cells pre-transfected with si-NC, si-circ-PREX1, si-circ-PREX1 and anti-miR-NC, or si-circ-PREX1 and anti-miR-140-3p. B, C Western blotting examined WNT5B protein level in IL-1β-induced C28/I2 cells pre-transfected with circ-PREX1, pcDNA, anti-miR-140-3p, or anti-miR-NC, and then treated with paeoniflorin (50 μM) for 4 h. *P < 0.05

Article Snippet: The primary antibodies targeting Bcl-2 (sc-7382), Bax (sc-7480), WNT5B (sc-376249), and GAPDH (sc-32233) were purchased from Santa Cruz (Shanghai, China), as well as the secondary antibody targeting mouse IgG (sc-516102).

Techniques: In Vitro, Western Blot, Transfection

Journal: International journal of oncology

Article Title: Depletion of JARID1B induces cellular senescence in human colorectal cancer.

doi: 10.3892/ijo.2013.1799

Figure Lengend Snippet: Figure 2. JARID1B controlled cell cycle and p16̸INK4A expression. (A) Cell cycle phase of Fucci‑labeled HCT116 cells separated by flow cytometry and FACS. (B) Relative JARID1B expression by stage (early and late G1, S and G2̸M). (C) ChIP assay of the promoter region of p16̸INK4A in Colo201 cells. (D) The relative ratio is shown in columns. me1, monomethyl; me2, dimethyl; me3, trimethyl. The control includes all methyl modifications.

Article Snippet: The sections were incubated with rabbit anti-JARID1B antibody (1:100; Novus Biologicals) overnight at 4 ̊C.

Techniques: Expressing, Flow Cytometry, Control

Journal: International journal of oncology

Article Title: Depletion of JARID1B induces cellular senescence in human colorectal cancer.

doi: 10.3892/ijo.2013.1799

Figure Lengend Snippet: Figure 1. Ubiquitous JARID1B expression in clinical samples and cell lines. (A) A sample of clinical colon adenocarcinoma (moderately differentiated); left panel, hematoxylin and eosin staining (x200); right panel, immunohistological staining of JARID1B (x200). Scale bar, 100 µm. (B) JARID1B expression in 11 CRC cell lines and one positive control (SK MEL). (C) Relative JARID1B expression in 11 CRC cell lines and one positive control (SK MEL). (D) Both synthesized oligo RNA‑mediated (siRNA) and lentiviral‑mediated depletion (shRNA) of endogenous JARID1B resulted in a significant increase in p16̸INK4A expression in Colo201 cells. Relative expression is shown.

Article Snippet: The sections were incubated with rabbit anti-JARID1B antibody (1:100; Novus Biologicals) overnight at 4 ̊C.

Techniques: Expressing, Staining, Positive Control, Synthesized, shRNA

Journal: International journal of oncology

Article Title: Depletion of JARID1B induces cellular senescence in human colorectal cancer.

doi: 10.3892/ijo.2013.1799

Figure Lengend Snippet: Figure 4. Tumorigenicity of JARID1B‑depleted cells. (A) Visualization of JARID1B promoter activity using fluorescent d2‑Venus labeling. (B) Quantitative RT‑PCR analysis of JARID1B in sorted endogenous JARID1B‑expressing and JARID1B‑non‑expressing CRC Colo201 cells (JPCs and JNCs, respectively). (C) Three weeks after injection, JPCs (blue circle) and JNCs (red circle) in mice. (D) Tumorigenicity of sorted endogenous JPCs or JNCs. JPCs had higher tumorigenicity.

Article Snippet: The sections were incubated with rabbit anti-JARID1B antibody (1:100; Novus Biologicals) overnight at 4 ̊C.

Techniques: Activity Assay, Labeling, Quantitative RT-PCR, Injection

Journal: International journal of oncology

Article Title: Depletion of JARID1B induces cellular senescence in human colorectal cancer.

doi: 10.3892/ijo.2013.1799

Figure Lengend Snippet: Figure 3. Induction of CRC cellular senescence by JARID1B depletion. (A) Phase‑contrast microscopy of SA‑β‑gal activity in JARID1B‑depleted and control Colo201 cells. NAC, N‑acetyl cysteine; H2O2, hydrogen peroxide. Scale bar, 100 µm. (B) Quantitative analysis of SA‑β‑galactosidase in Colo201 cells. (C) Intracellular ROS levels measured using CellROX‑labeled APC and flow cytometry in Colo201 cells. (D) Immunoblot analysis of SA proteins in JARID1B‑depleted and control Colo201 cells.

Article Snippet: The sections were incubated with rabbit anti-JARID1B antibody (1:100; Novus Biologicals) overnight at 4 ̊C.

Techniques: Microscopy, Activity Assay, Control, Flow Cytometry, Western Blot

Journal: International journal of oncology

Article Title: Depletion of JARID1B induces cellular senescence in human colorectal cancer.

doi: 10.3892/ijo.2013.1799

Figure Lengend Snippet: Figure 5. Suppression of cell growth and chemoresistance by JARID1B depletion. (A) Lentiviral‑mediated depletion of endogenous JARID1B suppressed Colo201 cell proliferation compared with that of the controls, as shown by MTT assay (proliferation assay). (B) Chemosensitivity assay. JARID1B‑depleted and control Colo201 cells were exposed to 5‑FU in an MTT assay (proliferation assay). (C) Flow cytometric analyses of endogenous JARID1B‑depleted Colo201 cells (#1 and #2). CD44+̸ALDH+ fractions were smaller in JARID1B‑depleted cells than in the control cells.

Article Snippet: The sections were incubated with rabbit anti-JARID1B antibody (1:100; Novus Biologicals) overnight at 4 ̊C.

Techniques: MTT Assay, Proliferation Assay, Control

Journal: ERJ Open Research

Article Title: Is MUC5B rs35705950 promoter polymorphism associated with chronic lung allograft dysfunction?

doi: 10.1183/23120541.00006-2025

Figure Lengend Snippet: Outcomes risks according to donors’ or recipients’ MUC5B polymorphism . Outcomes considered were antibody-mediated rejection, acute cellular rejection, restrictive or mixed allograft syndrome (logistic regressions), and chronic lung allograft dysfunction (Cox model). Presence of MUC5B polymorphism in recipients’ genotypes was significantly associated with the protection from antibody-mediated rejection (OR 0.24, 95% CI 0.07–0.65; p=0.01).

Article Snippet: Genotyping of the MUC5B rs35705950 polymorphism was performed using an allelic discrimination technique based on Taqman real-time quantitative PCR (assay ID C_1582254_20 Thermo Fisher Scientific), with two probes specific to each allelic variant.

Techniques:

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Western Blot, Recombinant, Derivative Assay, Control, Quantitative RT-PCR, Expressing, Glycoproteomics