aqp5 crispr activation plasmid  (Santa Cruz Biotechnology)

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Ex Vivo, CRISPR, Genome Wide, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: In Vivo, CRISPR, Ex Vivo

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling, Functional Assay

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: CRISPR, Knock-Out, In Vitro

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PGC-1α protects against MASH via Tim23-dependent inhibition of DRP1-mediated ferroptosis

doi: 10.1038/s41419-026-08493-8

Figure Lengend Snippet: Primary hepatocytes were transfected with negative control (NC) or PGC-1α CRISPR activation plasmid (PGC-1α CRISPR ACT) and treated with PA, leading to four groups of the Act-NC, Act-NC + PA, Act-PGC-1α and Act-PGC-1α + PA. A Oil Red O, Perls’ Blue staining and Tim23 Immunohistochemistry. B , B ’ Western blot analyses of the relative levels of hepatic PGC-1α, Tim23, Drp1, P-Drp1 Ser616 , ACSL4, GPX4 and FTH1 to GAPDH. C RT-qPCR analyses of the relative levels of PGC-1α, Drp1, ACSL4, GPX4, TFR1 and FTH1 mRNA transcripts. D The GSH levels. E Images of MitoTracker and MitoSOX staining. F Images of C11-BODIPY 581/591 staining. Data are representative images or expressed as the mean ± SD of each group ( n = 3) from at least three independent treatments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Special reagents included primary antibodies against Drp1, Nrf1, P-Drp1ser616, alpha-smooth muscle actin (α-SMA), collagen I, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c oxidase subunit 4 (COXIV) and cleaved caspase-3 (Cell Signaling Technology, Beverly, USA); PGC-1α, ACSL4, tumor necrosis factor (TNF)α, Hepatocyte nuclear factor 4-alpha (HNF-4α), Desmin and interleukin (IL)-6 (Abcam, Cambridge, USA); Tim23 (Santa, TX, USA); glutathione peroxidase 4 (GPX4), Ferroton heavy chain 1 (FTH1), transferrin receptor 1 (TFR1) and F4/80 (Proteintech, Wuhan, China); Lymphatic Vessel Endothelial Receptor-1(Lyve-1) (ABclonal, Wuhan, China); Nrf2, P-MLKL, GSDMD-N (HUABIO, Hangzhou, China); special kits for hematoxylin and eosin (H&E), Sirius Red, and Oil Red staining (Solarbio, Beijing, China); immunohistochemical staining kit (Maixin Biological Technology, Fujian, China); the kits for measurements of alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), glucose, glutathione (GSH), malondialdehyde (MDA), and iron contents (Jiancheng Biological Engineering Institute, Nanjing, China); enzyme-linked immunosorbent assay (ELISA) kits for measurements of insulin (Crystal Chem, Chicago, USA); Tissue mitochondria isolation kit (Beyotime, Shanghai, China); Perls’ blue (Sbjbio, Nanjing, China); PGC-1α clustered regularly interspaced short palindromic repeats (CRISPR) activation plasmid (sc-400070-ACT) and Protein A/G plus-agarose (Santa Cruz Biotechnology, CA, USA); PGC-1α and Drp1 small interfering RNAs (siRNAs) (LIKELI, Beijing, China); dual luciferase assay kits and TnT® quick coupled transcription/ translation systems (Promega, Wisconsin, USA); Lipofectamine 2000, C-11 BODIPY 581/591, MitoSOX and Mitotracker (Invitrogen, CA, USA); JC-1 (Chemodex, SG, SUI); Ferrostatin-1 (Fer-1) (MedChemExpress, NJ, USA).

Techniques: Transfection, Negative Control, CRISPR, Activation Assay, Plasmid Preparation, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: AKG-TET axis is central to senescence plasticity

doi: 10.1016/j.isci.2025.114298

Figure Lengend Snippet: AKG-TET deficiency leads to replicative senescence aHDFs (3 × 10 5 cells/mL) were seeded in each culture plate and were treated, in triplicate, without (UT) or treated (T) with C35 (5 μM), peptides (20 μM), TET1 siRNA ( TET1 i ; 300 nM). Cells treated with Bleomycin (Bleo; 5 μg/mL), or H 2 O 2 (100 μM) were used as positive control. Cells were incubated with 30 μM BrdU for 18 h prior to removal of both the cells and culture media following 7 days of treatment. (A) Heatmap of TET gene expression. (B) TET activity and global DNA levels of 5 mC, 5hmC, and 5 fC. (C) Expression patterns of energy/stress and nutrient-sensing pathways in aging PBMCs mirror those observed in young, actively replicating aHDFs (week 3) undergoing senescence, as well as those treated with TET1 i , C35, and RLS. (D) Expression level of hTERT , NAMPT , and PCNA . (E) Expression level of COL1A1 and ELN. (F) Cellular UPS activity, ATP, NAD + /NADH ratio, and LC3B levels. (G) Cellular levels of LC3B without (−) and after pre-treatment with (+) Bafilomycin A1 (1 μM) for 24 h. (H) Cellular ROS. (I) Extent of oxidative DNA damage assessed by the cellular level of 8OHdG. Positive control was comprised of cells treated with H 2 O 2 and Bleomycin (Bleo). Negative control was comprised of cells treated with CLV. (J) γH2AX immunolocalized to nuclei. For positive control, cells were treated with Bleomycin (Bleo). Intranuclear γH2AX (green) appears as focal spots (red arrows) or as a diffuse pan-nuclear pattern (yellow arrows) in nuclei marked by DAPI (blue) staining. Nuclei are further highlighted by double hashed lines. The boxes in the left pane (scale bars 10 μM) are magnified in the right insets (scale bars 2.5 μm). (K) NFKB1 gene expression. (L) Level of intra-nuclear phosphorylated p65 (p65P). (M) Cellular levels of ROS, IL6, and IL8 were secreted into culture media. (N) Expression levels of senescence markers ( CDKN2A , CDKN1A , CDKN1B , LTA4H , TIMP1 , and MMP1 ). (O) Expression level of LDH toxicity, SAβ-Gal activity, and extracellular level of lactic acid (LA). (P) LDHA1 gene expression. (Q) Rate of proliferation assessed by the quantitation of BrdU incorporated into the nuclei of cells in S-phase. Bar and line graphs show the means ± SD. Boxplots show the first and third quartiles and median values. Points are shown as empty and mean points as filled circles. The distribution of data points is shown by Beeswarm in Violin plots. Statistical significance was assessed using Student’s t test for two-group comparisons. p -values are presented as follows: ns (not significant), p ≤ 5 × 10 −1 , ∗p ≤ 5 × 10 −2 , ∗∗p ≤ 5 × 10 −3 , ∗∗∗p ≤ 5 × 10 −4 .

Article Snippet: CRISPR activation system for TET1 ( TET1 CR ) , Santa Cruz Biotechnology (Santa Cruz, CA) , sc-400845-ACT-2.

Techniques: Positive Control, Incubation, Gene Expression, Activity Assay, Expressing, Negative Control, Staining, Quantitation Assay

Journal: iScience

Article Title: AKG-TET axis is central to senescence plasticity

doi: 10.1016/j.isci.2025.114298

Figure Lengend Snippet: AKG-TET dependent resilience to damage and protection against damage-induced senescence Proliferating aHDFs (0.3 × 10 6 /mL) and PBMCs (70 years; PBMC 70Yr, 1 × 10 6 /mL) were seeded in culture plates and pre-treated in triplicate without (untreated: UT) or with H 2 O 2 (100 μM) for 24 h. After 24 h, cells were washed and treated without or with CLV (20 μM), or CRISPR TET1 ( TET1 CR ;1 μg/mL). Cells were incubated with 30 μM BrdU for 18 h prior to removal of both the cells and culture media following 7 days of treatment. (A) Expression levels of TETs in PBMCs. (B) AKG bioavailability in PBMCs. (C) Expression levels of energy/stress and nutritional sensors in PBMCs. (D) Heatmap of senescence marker gene expression in PBMCs. (E) Level of extracellular lactic acid (LA) and SAβ-Gal activity in PBMCs. (F) Quantification of BrdU and ROS (qROS) in PBMCs. Bar and line graphs show the means ± SD. Statistical significance was assessed using Student’s t test for two-group comparisons. p -values are presented as follows: ns (not significant), p ≤ 5 × 10 −1 , ∗p ≤ 5 × 10 −2 , ∗∗p ≤ 5 × 10 −3 , ∗∗∗p ≤ 5 × 10 −4 . Points are shown as empty and mean points as filled circles.

Article Snippet: CRISPR activation system for TET1 ( TET1 CR ) , Santa Cruz Biotechnology (Santa Cruz, CA) , sc-400845-ACT-2.

Techniques: CRISPR, Incubation, Expressing, Marker, Gene Expression, Activity Assay

Journal: iScience

Article Title: AKG-TET axis is central to senescence plasticity

doi: 10.1016/j.isci.2025.114298

Figure Lengend Snippet: AKG-TET deficient senescence state is reversible Young replicatively proliferating aHDFs (week 3, 0.3 × 10 6 /mL)) were seeded in culture plates and pre-treated in triplicates without (UT) and with C35 (5 μM), RLS (20 μM), or TET1 siRNA ( TET1 i ; 300 nM). Aliquot of cells were incubated with 30 μM BrdU for 18 h prior to removal of both the cells and culture media following 7 days of treatment. The remaining cells were re-seeded in equal numbers and cultured without treatment for an additional 7 days (withdrawal). Cultures were incubated with 30 μM BrdU for 18 h prior to removal of both the cells and culture media. (A) Quantification of TET activity, and 5 mC and 5fc levels. (B) Expression level of energy/stress and nutritional sensor genes. (C) Expression level of NFKB1 , RELA , IKBA , COL1A1, and ELN . (D) γH2AX immunolocalized (cyan) in nuclei. Nuclei stained for DAPI (deep blue) are marked by double hashed lines. (E) Extracellular level of lactic acid (LA), SAβ-Gal activity, LDH toxicity, and UPS. (F) Intracellular level of ROS and 8OHdG. (G) Heatmap of senescence markers and RRM2 gene expression. (H) Rate of proliferation assessed by BrdU incorporation into the nuclei of cells in S-phase. Bar and line graphs show the means ± SD. Boxplots show the first and third quartiles and median values. All points are shown as empty, and mean points as filled circles. The distribution of all data points is shown by Beeswarm in Violin plots. Statistical significance was assessed using Student’s t test for two-group comparisons. p -values are presented as follows: ns (not significant), p ≤ 5 × 10 −1 , ∗p ≤ 5 × 10 −2 , ∗∗p ≤ 5 × 10 −3 , ∗∗∗p ≤ 5 × 10 −4 .

Article Snippet: CRISPR activation system for TET1 ( TET1 CR ) , Santa Cruz Biotechnology (Santa Cruz, CA) , sc-400845-ACT-2.

Techniques: Incubation, Cell Culture, Activity Assay, Expressing, Staining, Gene Expression, BrdU Incorporation Assay

Journal: iScience

Article Title: AKG-TET axis is central to senescence plasticity

doi: 10.1016/j.isci.2025.114298

Figure Lengend Snippet: Activation of the AKG-TET axis reverses replicative and age induced senescence Replicatively induced senescence. Replicatively senescent aHDFs (0.3 × 10 6 cells/mL) were seeded in triplicate in each plate and were treated without (UT) and with RLS (20 μM), CLV (20 μM), or CRISPR TET1 plasmids (TET1 CR , 1 μg/mL). Cells were incubated with 30 μM BrdU for 18 h prior to removal of both the cells and culture media following 7 days of treatment. (A) Expression level of TET s. (B) AKG bioavailability and TET activity (TET act ). (C) Expression level of energy/stress and nutritional sensor gene expression. (D) Intracellular level of ROS. (E) Heatmap of senescence marker and RRM2 gene expression. (F) Level of lactic acid (LA) in culture media. (G) Cellular SAβ-Gal activity. (H) Quantitation of nuclear BrdU. (I) Total cell numbers. Age induced senescence. PBMCs (PBMC 70Yr ) were seeded (1 × 10 6 cells/mL) in triplicate in each well of a 24-well plate and were treated without (UT) and with RLS (20 μM), CLV (20 μM), or CRISPR TET1 plasmids ( TET1 CR : 1 μg/mL). Cells and culture media were removed on day 7 of treatment for analysis. (J) Quantitation of bioavailable AKG and TET activity in cells treated with RLS versus CLV. (K) qPCR quantitation of the expression level of TET s. (L) qPCR quantitation of the expression level of energy/stress and nutritional sensor gene expression. (M) Quantitation of BrdU incorporated into nuclei of cells in S-phase, lactic (LA) acid in culture media, cellular SAβ-Gal activity, and ROS. (N) Heatmap of senescence markers and RRM2 in PBMCs. Bar and line graphs show the means ± SD. Boxplots show the first and third quartiles and median values. All points are shown as empty, and mean points as filled circles. The distribution of all data points is shown by Beeswarm in Violin plots. Statistical significance was assessed using Student’s t test for two-group comparisons. p -values are presented as follows: ns (not significant), p ≤ 5 × 10 −1 , ∗p ≤ 5 × 10 −2 , ∗∗p ≤ 5 × 10 −3 , ∗∗∗p ≤ 5 × 10 −4 , ∗∗∗∗p ≤ 5 × 10 −5 .

Article Snippet: CRISPR activation system for TET1 ( TET1 CR ) , Santa Cruz Biotechnology (Santa Cruz, CA) , sc-400845-ACT-2.

Techniques: Activation Assay, CRISPR, Incubation, Expressing, Activity Assay, Gene Expression, Marker, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Triglyceride secretion rate and analyses of secreted lipoproteins following tyloxapol administration in rats . ( A ) Rate of plasma triglyceride concentration measured after a 16 h fast followed by intraperitoneal injection of tyloxapol (700 mg per kg body weight) in male wild-type and homozygous Cyp7b1 -deficient rats (n = 11 per group). * p < 0.05 according to Mann–Whitney’s U-test. FPLC separation of plasma lipoproteins from both groups of rats four hours after tyloxapol injection and their APOB contents are reflected in panel ( C ). Their triglyceride and cholesterol contents are depicted in ( B , D ), respectively. Open squares correspond to wild-type and black triangles to Cyp7b1 -deficient rats.

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Concentration Assay, Injection

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Hepatic analyses of rats after 16 h of fasting. ( A ) Representative Western blot analysis of plasma membrane proteins related to lipoprotein metabolism. Hepatic plasma membrane contents of ( B ) low-density lipoprotein receptor (LDLR); ( C ) ATP-binding cassette subfamily A member 1 (ABCA1); ( D ) low-density lipoprotein receptor-related protein 1 (LRP1); and ( E ) syndecan 1 (SDC1). Results of Western blot analyses were normalized to the data of flotillin 2 (FLOT2) as loading control. ( F ) Ldlr mRNA and ( G ) Pcsk9 mRNA expressions. Values are reported as relative arbitrary unit (AU) levels obtained by RT-qPCR and normalized to Rn18s. ( H ) Hepatic levels of 25-hydroxycholesterol. ( I ) Morphometric changes in lipid droplet area expressed as a percentage of the total liver section. Data are shown as means ± SD with their individual values of 4 WT and 6 homozygous Cyp7b1 -deficient (KO) rats. Statistical analysis was performed using the Mann–Whitney U-test. * p < 0.05, ** p < 0.02, *** p < 0.01, and **** p < 0.001 vs. control.

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Western Blot, Clinical Proteomics, Membrane, Binding Assay, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Postprandial analyses of male rats eight hours after receiving a fat bolus . ( A ) 16 h fasted male rats received an intragastrical administration of 5 mL of virgin olive oil (16 mL kg −1 ). Differential values of plasma TG eight hours after the oral gavage minus those from basal values are shown as individual, mean, and standard deviation values for wild-type (WT, n = 8) and homozygous KO (n = 17). Statistical analysis was performed using the Mann–Whitney U test. * p < 0.05 and *** p < 0.001 vs. control. Plasma lipoproteins were separated by FPLC and their APOA4 contents are reflected in panel ( B ). Their cholesterol and phosphatidylcholine (PC) contents are depicted in ( C , D ), respectively. Open squares correspond to wild-type and black triangles to homozygous Cyp7b1 -deficient rats. ( E ) Hepatic levels of 25-hydroxycholesterol of rats sacrificed eight hours after the oral gavage. ( F ) Morphometric changes in the lipid droplet area expressed as a percentage of the total liver section. ( G ) Association between hepatic Abca1 expression and levels of 25-hydroxycholesterol. Spearman’s ρ coefficient and significance are shown.

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Standard Deviation, MANN-WHITNEY, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Differentially expressed genes according to Cyp7b1 deficiency in rats. ( A ) Hepatic Cyp7b1 mRNA expression in WT (n = 8), homozygous Cyp7b1 -deficient (KO, n = 10), and homozygous Cyp7b1 -deficient (n = 4) rats rescued by hydrodynamic expression of pLIVE- Cyp7b1 (rescued KO) using RT-qPCR. ( B ) Venn diagram analysis showing the significant transcripts (false discovery rate < 0.01) among the different groups using RNAseq from the livers. ( C ) Hepatic expression of Fasn , Igfbp2 , and Pcsk9 normalized to Rn18s using RT-qPCR. ( D ) Significant association among gene expressions. Spearman’s ρ correlation coefficients are shown. ( E ) Western blot analysis of hepatic protein expressions of the non-glycosylated IGFBP2 isoform of 36 kDa normalized to ACTIN ( left ) and of the glycosylated IGFBP2 isoform of 50 kDa normalized to ACTIN ( right ). ( F ) Plasma protein content of PSK9 (74 kDa) normalized to total protein loaded into the gel. Data are shown as individual, means ± SD. Statistical analysis was performed using the Mann–Whitney U-test. ** p < 0.02 and *** p < 0.01 vs. control. ( G ) Ldlr and ( H ) Mylip expressions normalized to Rn18s using RT-qPCR. Data are means ± SD of arbitrary units. Statistical analysis was performed using one-way ANOVA and Tukey’s post hoc test. * p < 0.05, ** p < 0.02, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Clinical Proteomics, MANN-WHITNEY, Control

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Characterization of a stable human CYP7B1 -knockout HepG2 cell line. ( A ) Levels of oxysterols: 4β-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. ( B ) Gene expressions of FASN , IGFBP2 , and PCSK9 normalized to PPIB in wild-type (WT) and CYP7B1 -knockout (KO) HepG2 cells. ( C ) FASN , IGFBP2 , and PCSK9 gene expressions of wild-type HepG2 cells incubated with 250 nM 4β-hydroxycholesterol (4β-HC), 25-hydroxycholesterol (25-HC), or 27-hydroxycholesterol (27-HC) for 24 h. ( D ) Gene expressions of CYP7B1 -KO HepG2 cells incubated with 250 nM 4β-hydroxycholesterol (4β-HC), 25-hydroxycholesterol (25-HC), or 27-hydroxycholesterol (27-HC) for 24 h. Data are shown as individual, means ± SD of four replicates except for 4β-hydroxycholesterol (eight replicates). Statistical analysis was performed using one-way ANOVA and Tukey’s post hoc test or Mann–Whitney’s U test. * p < 0.05 and ** p < 0.02.

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Knock-Out, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Disruption of Cyp7b1 Controls IGFBP2 and Prediabetes Exerted Through Different Hydroxycholesterol Metabolites

doi: 10.3390/ijms262411994

Figure Lengend Snippet: Summary of phenotype of Cyp7b1 deficiency in rats. The absence of this enzyme increases levels of 25-HC and through this, broad metabolic control is exerted on FASN, IGFBP2, PCSK9, LDLR, SCD1, ABCA1, and LRP1. Created in Biorender.com, Osada (2025), https://app.biorender.com/illustrations/67ddc3c130709ae66c1676b6 .

Article Snippet: Then, the medium was withdrawn, and the cells were washed twice with PBS before being transfected with CYP7B1 HDR and CYP7B1 CRISPR/Cas9 KO plasmids (Santa Cruz Biotechnology) using lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on INSR/IGF1R and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R and INSR knockdown on the Pas2r12-mediated cytosolic delivery of EGFP. Western blot analyses ( A – C ) and confocal laser scanning microscopy images ( D , E ). Graphs B and C show IGF1R and INSR expression levels, respectively, normalized to GAPDH. Cells analyzed with confocal laser scanning microscopy in panel A were analyzed by Western blot ( A – C ). ( D ) Pas2r12-mediated cytosolic delivery of EGFP in knockdown cells, with EGFP fluorescence shown in green, and ( E ) percentage of cells exhibiting cytosolic EGFP delivery. Scale bars represent 20 μm. Statistical comparisons were performed against siNC cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Assessment of IGF1R overexpression. ( A ) Verification of IGF1R expression levels in HEKI cells. ( B ) Relative levels of IGF1R expression normalized to GAPDH based on the data in panel A. Values for IGF1R/GAPDH are expressed compared with parental HEK293 cells (set to 1). Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3. ( C ) Subcellular localization of IGF1R is shown in green; nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 20 μm.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing the cellular uptake of Pas2r12–EGFP in IGF1R-overexpressing (HEKI) cells. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in HEKI cells compared with parental HEK293 cells (set to 100%). Merged images show EGFP fluorescence (green) and differential interference contrast. Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Solvent, Control