Journal: Brain : a journal of neurology

Article Title: A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

doi: 10.1093/brain/awy126

Figure Lengend Snippet: Figure 4 Sub-retinal injections in adult mice show superior transduction ability of AAV-TT throughout different layers of the retina. GFP expressing AAV2 and AAV-TT were injected contralaterally in adult mice at a dose of 2 109 vg per eye (n = 4 per condition). (A) Representative confocal z-projections of transverse sections of the retina taken 4 weeks after sub-retinal injections show that AAV-TT targets a higher number of RPE and photoreceptor cells and mediates increased transgene expression as compared to AAV2. (B) AAV-TT cannot efficiently penetrate the retina when injected via the intra-vitreal route; intra-vitreal injection of AAV2 leads to transduction of retinal ganglion cells. GC = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; PS = photoreceptor segments; RPE = retinal pigment epi- thelium. Scale bar = 200 mm.

Article Snippet: The AAV-CAG coHGSNAT transgene plasmid was constructed by replacing the GFP coding sequence in the pTRUF-11 plasmid (ATCC, MBA-331) with a human codon-optimized HGSNAT cDNA (including a Kozak sequence) into the SbfI and SphI sites.

Techniques: Transduction, Expressing, Injection

Journal: Brain : a journal of neurology

Article Title: A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

doi: 10.1093/brain/awy126

Figure Lengend Snippet: Figure 6 AAV-TT effectively corrects disease phenotype in MPSIIIC mice. (A) AAV-HGSNAT vectors, packaged into AAV9 or AAV-TT capsids, were used to treat MPSIIIC mice. (B) Schematic of injection site and brain sectioning used for enzyme level determination. (C) Overview of treatment and analysis scheme. (D) Hyperactivity as measured by distance travelled by wild-type (wt, n = 8) and MPSIIIC sham (n = 11), AAV9 (n = 8) and AAV-TT (n = 7) treated MPSIIIC mice. (E) Configuration of Y-maze. (F) Cognitive ability as measured by the percentage of alternation in the Y- maze of wild-type (n = 10) and MPSIIIC sham (n = 12), AAV9 (n = 9) and AAV-TT (n = 9) treated MPSIIIC mice. (G) Vector genome copy numbers (vg/cell) measured by qPCR in whole brain tissue preparations from wild-type (n = 1), MPSIIIC (n = 1), AAV9 (n = 6) and AAV-TT (n = 6) treated MPSIIIC mice. (H) HGSNAT enzyme activity measured in whole brain preparations of wild-type (n = 6), MPSIIIC (n = 6), AAV9 (n = 6) and AAV-TT (n = 6) treated MPSIIIC mice. (I) HGSNAT enzyme activity measured in brain sections R1–R5. (J) Total IgG antibody responses against AAV9 capsid proteins as measured by ELISA. Absence of capsid-specific antibodies in brain homogenates of AAV9-coHGSNAT (n = 6) treated mice. (K) Total IgG antibody responses against AAV-TT capsid proteins as measured by ELISA. Absence of capsid-specific antibodies in brain homogenates of AAV-TT-HGSNAT (n = 6) treated mice. Positive controls consist of mice treated with vectors and adjuvant. ANOVA followed by Tukey’s post hoc multiple comparison test. Data are presented as mean SEM; *P 5 0.05; **P 5 0.01; ***P 5 0.001; ****P 5 0.0001.

Article Snippet: The AAV-CAG coHGSNAT transgene plasmid was constructed by replacing the GFP coding sequence in the pTRUF-11 plasmid (ATCC, MBA-331) with a human codon-optimized HGSNAT cDNA (including a Kozak sequence) into the SbfI and SphI sites.

Techniques: Injection, Plasmid Preparation, Activity Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Comparison

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: a ATGL −/− HeLa cells transfected with FLAG-PNPLA3 were treated overnight with (from left to right) 250 μM of OA, LA, α-LA, AA (250 μM), or DHA (125 μM) + EPA (125 μM), and then immunostained with a FLAG antibody. LDs were stained with BODIPY. b Quantification of LD area in ( a ). c ATGL −/− HeLa cells transfected with FLAG-tagged (from left to right) PNPLA3-WT, PNPLA3-S47A, or PNPLA3-I148M were treated overnight with 250 μM LA and then imaged as in ( a ). d Quantification of LD area in ( c ). e ATGL −/− HeLa cells transfected with Myc-tagged PNPLA3 or co-transfected with Flag-G0S2 were treated overnight with 250 μM LA and then imaged as in ( a ). f Quantification of LD area in ( E ). g WT control (left) or CGI-58 −/− (right) Huh7 cells transfected with FLAG-PNPLA3 were treated with 250 μM LA overnight and then imaged as in ( a ). h Quantification of LD area in ( g ). i PNPLA3 −/− primary mouse hepatocytes were isolated and infected with null, PNPLA3 WT or PNPLA3 I148M expressing adenovirus, and loaded with 250 μM OA + 3 H-OA (left) or 250 μM LA + 14 C-LA (right). After cell lysis, isolated LDs were normalized for total TG content and incubated at 37 °C with 20 μM ATGListatin in a 5% FA-free BSA solution. At the indicated intervals, fractional aliquots were removed from the reaction mixture, and albumin-bound FA was extracted for scintillation counting ( n = 4 per condition from a representative experiment). Scale bar in microscopic images = 10 μm. Error bars in ( i ) represent mean ± SD. (*) indicates statistical significance between [Null] and [PNPLA3 WT], (#) indicates statistical significance between [PNPLA3 WT] and [PNPLA3 I148M], and (+) indicates statistical significance between [Null] and [PNPLA3 I148M]. Box-plot elements: center line, median; box limits, upper and lower quartiles. * p < 0.05, ** P < 0.01, *** p < 0.001, **** p < 0.0001, two-sided t test. Specific p values: b Untransfected+OA vs PNPLA3 + OA: p = 0.488; Untransfected+OA vs Untransfected+LA: p = 0.961; Untransfected+LA vs PNPLA3 + LA: p = 1.22E-08; Untransfected+LA vs PNPLA3+aLA: p = 1.35E-06; Untransfected+LA vs PNPLA3 + AA: p = 2.25E-08; Untransfected+LA vs PNPLA3 + DH/EPA: p = 1.59E-08. d PNPLA3 WT + LA vs PNPLA3 S47A + LA: p = 2.46E-05; PNPLA3 WT + LA vs PNPLA3 I148M + LA: p = 6.43E-04; PNPLA3 S47A + LA vs PNPLA3 I148M + LA: p = 0.237. f p = 9.60E-06. h p = 9.22E-04. i OA loaded LDs: Null vs PNPLA3 WT/2 h: p = 0.0235, 4 h: p = 0.0011, 6 h: p = 0.030; Null vs PNPLA3 I148M/2 h: p = 0.458, 4 h: p = 0.804, 6 h: p = 0.715; PNPLA3 WT vs PNPLA3 I148M/2 h: p = 0.0060, 4 h: p = 0.00051, 6 h: p = 0.0538; LA loaded LDs: Null vs PNPLA3 WT/2 h: p = 3.63E-05, 4 h: p = 8.38E-04, 6 h: p = 3.91E-08; Null vs PNPLA3 I148M/2 h: p = 0.708, 4 h: p = 0.885, 6 h: p = 0.0029; PNPLA3 WT vs PNPLA3 I148M/2 h: p = 2.68E-06, 4 h: p = 6.87E-05, 6 h: p = 6.02E-07.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Transfection, Staining, Isolation, Infection, Expressing, Lysis, Incubation

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: 10-week-old WT or ATGL −/− mice fed a chow diet were injected with Ad-null or Ad-FLAG-PNPLA3. Tissues were collected following a 12-h fast ( n = 8/group). a Immunoblotting analysis of ATGL and FLAG-PNPLA3 expression in the liver. b Total liver TG content. c Total plasma TG content. d – j Shotgun lipidomic analysis of whole liver tissue ( n = 3/group). d Heatmap indicating fold-change of individual TG species relative to control (WT mice injected with Ad-null) arranged by increasing number of double bonds. Individual FA composition of TGs presented as absolute FA abundance ( e ) and as percentage of the total FA pool ( f ). Abundance of total PLs ( g ) and major PL classes ( h ). Absolute FA abundance in PE ( i ) and PC ( j ) species. All error bars represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001, two-sided t test. Specific p values: b (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.938; (Ad-null) ATGL + /+ vs ATGL −/−: p = 9.01E-07; (Ad-PNPLA3) ATGL + /+ vs ATGL −/−: p = 0.356; (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 1.09E-07. e 16:0, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0149; 16:1, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0228; 18:0, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.0176, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0156; 18:1, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0418; 18:2, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 4.33E-04, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00483; 18:3, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00876; 20:4, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0117. f 16:0, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.0211, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.831; 18:0, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0041, 18:1, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.126, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.581; 18:2, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.0186, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0297; 18:3, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0104. g (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.383; (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.000223. h PE, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.188, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00170; PC, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.713, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00934. i 16:0, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.158, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00356; 18:0, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.324, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00709; 18:2, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.000145; 22:6(ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.577, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0037. j 16:0, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.908, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.00154; 18:2, (ATGL + /+) Ad-null vs Ad-PNPLA3: p = 0.841, (ATGL−/−) Ad-null vs Ad-PNPLA3: p = 0.0105.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Injection, Western Blot, Expressing

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: a Experimental scheme (Biorender) for creation and treatment of PNPLA3-LKO mice. 8–10-week-old PNPLA3 fl/fl mice injected with either AAV-TBG-Null ( n = 8) or AAV-TBG-Cre ( n = 10) were fed COWD and given access to fructose/glucose in drinking water for 6 weeks. b Liver PNPLA3 mRNA expression as analyzed by qPCR. c Gross appearance of liver (scale bar = 1 cm). d Hematoxylin and eosin (H&E) staining of liver sections. e Total liver TG content. f Total plasma TG content. g Liver mRNA expression of lipogenic genes by qPCR. h Liver mRNA expression of ER stress response genes by qPCR. i Immunoblotting analysis of indicated lipolysis, lipogenesis, and ER-stress associated proteins in liver. j Experimental scheme (Biorender) for PNPLA3 ASO experiment. 10-week-old WT mice fed COWD and given fructose drinking water were treated with control or PNPLA3 ASO biweekly for 3 weeks ( n = 9/group). k Liver PNPLA3 mRNA expression in ASO mice by qPCR. l Total liver TG content. m Total plasma TG content in ASO mice. Data presented in ( e – h ) and ( k – m ) are independent replicates derived from individual mice. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, two-sided t test. Specific p values: e p = 0.00410. f p = 0.0443. k p = 7.73E-06. l p = 0.0389. m p = 0.0272.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Injection, Expressing, Staining, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: 10-week-old WT mice fed COWD were treated with control or PNPLA3 ASO for 3 weeks. Both groups were treated with the LXR agonist T09 prior to tissue collection ( n = 7/group). a Experimental scheme (Biorender). b Liver PNPLA3 mRNA expression. c Total liver TG content. d Total plasma TG content. e – k Shotgun lipidomics was performed on whole liver tissue ( n = 3/group). Individual FA species composition of TG presented as absolute FA abundance ( e ) and as percentage of the total FA pool ( f ). g Percentage of total FA pool in DAG. Abundance of total PLs ( h ) and major PL classes ( i ). j Total abundance of SFA, MFA, and PUFA acyl chains in PC. k Absolute FA abundance in PC. Data presented as mean ± SD * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001, two-sided t test. Specific p values: c p = 0.00211. d p = 0.0402. e 14:0, p = 7.24E-04; 16:0, p = 0.0160; 18:0, p = 0.00681; 20:0, p = 0.00296; 16:1, p = 0.00742; 18:1, p = 0.0166; 20:1, p = 0.0101; 18:2, p = 0.00998; 18:3, p = 0.0110; 20:3, p = 1.06E-04; 22:3, p = 8.61E-04; 20:4, p = 4.13E-04; 20:5, p = 0.00136; 22:5, p = 0.00113; 22:6, p = 0.00114. f 18:1, p = 0.509; 18:2, p = 0.0460. g 18:1, p = 0.00226; 16:2, p = 0.0310; 18:2, p = 0.0418. h p = 0.0214. i PC, p = 0.0459. j SFA, p = 0.186; MUFA, p = 0.236; PUFA, p = 0.0108. k 16:0, p = 0.805; 18:0, p = 0.114; 18:1, p = 0.192; 18:2, p = 0.327; 18:3, p = 0.00367; 20:3, p = 0.140; 20:4, p = 6.46E-04; 22:5, p = 0.0104; 22:6, p = 2.96E-04.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Expressing

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: a Representative time course of hepatic TG secretion in mice upon administration of Poloxomer-407. Priorly, 10-week-old WT mice were fed COWD and treated with control ( n = 6) or PNPLA3 ASO ( n = 7) and administered T09 for 3 weeks. b Data from ( a ) represented as secretion rate per hour. c – f Shotgun lipidomics was performed on plasma samples from the 3 h timepoint in ( a ) ( n = 4/group). Individual FA species composition of TG presented as absolute FA abundance ( c ) and as percentage of the total FA pool ( d ). Individual FA species composition of PC presented as absolute FA abundance ( e ) and as percentage of the total FA pool ( f ). Plasma samples pooled from control or PNPLA3 ASO mice administered T09 were fractionated by FPLC (4-5 mice/group). Lipoprotein fractions were measured for the content of TG ( g ) and cholesterol ( h ). i Plasma samples were immunoblotted for ApoB-100 and ApoB-48. j EM analysis of lipoprotein particles from control and PNPLA3 ASO mice +T09. k Quantification of VLDL particle size distribution of ( J ). Scale bar = 200 nm. Data presented as mean ± SD * p < 0.05, ** p < 0.01, two-sided t test. Specific p values: ( a ) 1 h, p = 0.214; 2 h, p = 0.00661; 3 h, p = 0.00544; 4 h, p = 0.00643. ( b ) p = 0.00212. ( c ) 16:0, p = 0.00923; 18:0, p = 0.0452; 16:1, p = 0.00309; 18:1, p = 0.00587; 18:2, p = 0.00125. e 16:0, p = 0.00718; 18:0, p = 0.0344; 18:1, p = 0.0109; 18:2, p = 0.00194; 20:3, p = 0.0456; 20:4, p = 0.00966; 22:6, p = 0.00454.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques:

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: a qPCR of PNPLA3 expression in primary hepatocytes transfected with control or PNPLA3 ASO ( n = 3/group). b BODIPY staining of LDs in cells after 4 h of lipid deprivation in high glucose medium following treatment with 250 μM OA or LA in low glucose medium. c Quantification of LD area in ( b ). Primary hepatocytes were pretreated with a mixture of unlabeled 125 μM OA + 125 μM LA ( n = 8/ASO group). Following 24 h in FA-free media, lipids were extracted from cells ( d ) and conditioned media ( e ) for quantitative TG analysis. Primary hepatocytes were pretreated with a mixture of unlabeled 125 μM OA + 125 μM LA containing either 3 H-OA or 14 C-LA. After a 24-h chase period, cellular and secreted lipids were analyzed for the ratio of 3 H-OA ( f ) or 14 C-LA ( g ) in PL vs TG ( n = 4/group). Primary hepatocytes were pretreated with either 125 μM of unlabeled OA containing 3 H-OA ( h ) or 125 μM of unlabeled LA containing 14 C-LA ( i ). After a 24 h chase period, secreted lipids were analyzed for ratio of radioactivity in PL vs.TG ( n = 3/group). Scale bar = 50 μm. Box-plot elements: center line, median; box limits, upper and lower quartiles. Data presented as mean ± SD * p < 0.05, ** p < 0.01, **** p < 0.0001, two-sided t test. Specific p values: c (Ctrl ASO) OA vs LA, p = 2.23E-06; (PNPLA3 ASO) OA vs LA, p = 0.177; (OA) Ctrl ASO vs PNPLA3 ASO, p = 0.00239; (LA) Ctrl ASO vs PNPLA3 ASO, p < 1E-10. d p = 0.00213. e p = 0.00666. f Cellular, p = 0.00775; Secreted, p = 0.783. g Cellular, p = 0.00437; Secreted, p = 0.0300. i p = 0.0424.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Expressing, Transfection, Staining, Radioactivity

Journal: Nature Communications

Article Title: PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein

doi: 10.1038/s41467-024-49224-x

Figure Lengend Snippet: a TG secretion from primary hepatocytes isolated from WT mice, infected with control, PNPLA3 WT or PNPLA3 I148M expressing adenovirus, and treated with 250 μM 1:1 OA:LA and 25 μM glycerol containing 3 H-glycerol ( n = 3/group). b–h 10-week-old WT mice fed a chow diet were injected with either Ad-null or Ad-PNPLA3-I148M. Tissues were collected following a 12-h fast for lipid ( n = 8/group) and lipidomic analysis ( n = 3/group). b Total liver TG content. Individual FA species composition of TG presented as absolute FA abundance ( c ) and as percentage of the total FA pool ( d ). e Abundance of total PLs. f Abundance of major PL classes. Individual FA species composition of PE presented as absolute FA abundance ( g ) and as percentage of the total FA pool ( h ). Individual FA species composition of PC presented as absolute FA abundance ( i ) and as percentage of the total FA pool ( j ) . k TG secretion from primary hepatocytes isolated from WT or PNPLA3-I148M-KI mice following treatment with 250 μM 1:1 OA:LA and 25 μM glycerol containing 3 H-glycerol ( n = 6/group). l–o 9-12-wk-old WT ( n = 10) and I148M-KI ( n = 7) mice were fed COWD and given access to fructose/glucose in drinking water for 3 weeks. l Total liver TG content. m Total plasma TG content. n Representative time course of hepatic TG secretion in WT ( n = 10) and PNPLA3-I148M-KI ( n = 7) mice upon administration of Poloxomer-407 ( o ) Data from ( n ) represented as secretion rate per hour. All error bars represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001, two-sided t test. Specific p values: a p = 0.0445. b p = 2.16E-04. c 14:0, p = 8.65E-04; 16:0, p = 5.10E-04; 16:1, p = .00215; 18:0, p = 1.75E-04; 18:1, p = 2.91E-04; 18:2, p = 2.36E-05; 18:3, p = 1.89E-04; 20:4, p = 0.434. d 14:0, p = 0.00386; 16:0, p = 0.6.23E-04; 18:1, p = 0.573; 18:2, p = 0.611; 18:3, p = 0.0700; 20:4, p = 0.00793. e p = 0.0406. f PE, p = 0.0487; PC, p = 0.0258. g 16:0, p = 0.0801; 18:2, p = 0.00376; 20:4, p = 0.00670. h 18:2, p = 0.0277; 20:3, p = 0.0618; 20:4, p = 0.114; 22:5, p = 0.0110. i 16:0, p = 0.00707; 18:1, p = 0.126; 18:2, p = 0.00415; 20:4, p = 0.021. j 18:0, p = 0.00761; 18:2, p = 3.43E-06; 20:4, p = 0.0429; 22:5, p = 0.00981. k p = 2.96E-04. l p = 1.60E-04. m p = 0.0223. n 1 h, p = 0.731; 2 h, p = 0.0269; 3 h, p = 4.19E-04. o p = 0.000522.

Article Snippet: Recombinant adenovirus encoding N-terminally FLAG-tagged mouse PNPLA3 under the control of a cytomegalovirus (CMV) promoter (Ad-FLAG-PNPLA3) was custom generated by Vector Biolabs.

Techniques: Isolation, Infection, Expressing, Injection

Journal: Cell Metabolism

Article Title: Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1

doi: 10.1016/j.cmet.2023.02.004

Figure Lengend Snippet:

Article Snippet: To ablate Cited1 , adeno-associated viruses carrying the Cre recombinase (AAV2/1-EF1a-EGFP-T2A-iCre, Vector Biolabs, Cat. No. VB2069) or control viruses carrying GFP (AAV2/1-EF1a-EGFP, Vector Biolabs, Cat. No. VB2084) were injected bilaterally (0.5 μl/side; 1x10 13 viral genomes/ml) into the MBH of Cited1 loxP/loxP mice (8-wk-old), as specified, using a motorized stereotaxic system from Neurostar (Tubingen, Germany).

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, RNAscope, Multiplex Assay, Diagnostic Assay, Gene Expression, Software

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Regulation of LPCAT3 mRNA expression by PPARδ activation. A, HepG2 cells were seeded in a 12-well cell culture plate. After 24 h, cells were transduced with Ad-shLacZ (50 MOI) or Ad-shPPARδ (50 MOI) adenovirus in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 48 h. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. (error bars) of two RNA samples with triplicate measurement per RNA sample. **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. B, HepG2 cells were transduced with Ad-GFP (50 MOI) or Ad-PPARδ (50 MOI) adenoviruses in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 24 h and then subsequently treated with 1 μm GW0742 or DMSO. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. of two RNA samples with triplicate measurement per RNA sample. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. C, real-time PCR quantification of LPCAT3 mRNA expression from HepG2, Huh7, and Hepa 1-6 cells treated with GW0742 (1 μm), L165041 (20 μm), or DMSO for 24 h. LPCAT3 expression levels were normalized to GAPDH mRNA levels, where the relative expression of LPCAT3 mRNA in DMSO-treated cells was set at 1. D and E, qRT-PCR quantification of LPCAT3 or ACSL4 mRNA expression from HepG2 cells treated with L165041 at the indicated concentrations. Bars, mean ± S.E. of triplicate measurements of each RNA sample. The data shown are representative of two independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with DMSO-treated samples. F, C57BL/6J mice fed a normal chow diet were injected with Ad-sh-mPPARδ (n = 4) or Ad-shLacZ (n = 4). Ten days after injection, mice were sacrificed for liver tissue collection. qRT-PCR was used to determine the relative expression levels (mean ± S.E., n = 4/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.001, compared with the vehicle group, which was set at 1.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Cell Culture, Transduction, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Up-regulation of human LPCAT3 promoter activity in HepG2 cells by PPARδ agonists and LXR agonist. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter construct. B, relative luciferase activities from HepG2 cells transfected with pLPCAT3-Luc promoter luciferase plasmid or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4–6 replicates/treatment and are expressed as ratio of luciferase/β-gal activity from each sample, where the relative luminescence from cells transfected with promoterless pGL3-basic vector and treated with DMSO is set to 1. *, p < 0.05 compared with DMSO-treated samples. The data shown are representative of three separate transfection experiments.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Mapping the functional PPRE site in human LPCAT3 promoter. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter constructs of wild type and PPRE mutants. B, relative luciferase activities from control and L165041-treated Huh7 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E.) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. ***, p < 0.001 compared with DMSO-treated samples. C, after transfection, cells were treated with DMSO as control, 1 μm GW3965, 10 μm L165041, or GW3965 + L165041 for 24 h before cell lysis for the luciferase and β-gal activity assay. The graph represents relative luciferase activities from control and treated HepG2 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. *, p < 0.05; ***, p < 0.001 compared with DMSO-treated samples. The data shown are representative of two separate transfection experiments.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Functional Assay, Luciferase, Construct, Control, Transfection, Plasmid Preparation, Activity Assay, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: EMSA and ChIP analyses of PPARδ association with PPRE1 site of LPCAT3 promoter in vitro and in vivo. A, chemiluminescent signal from cross-linked nylon membrane, where biotin-5′-end-labeled LPCAT3-PPRE1 (lanes 1–4) was incubated with 200 ng of PPARδ and 100 ng of RXRα recombinant proteins in the absence (lane 2) or presence of a 100-fold molar excess of unlabeled wild type probe (lane 3) or mutated probe (lane 4). B, ChIP analysis on HepG2 cells treated with DMSO or L165041 (10 and 20 μm), where rabbit anti-PPARδ or isotype IgG antibody-immunoprecipitated DNA samples and input DNA samples were PCR-amplified with primers specific for the LPCAT3-PPRE1 and LPCAT3-PPRE2 promoter region. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. C, real-time quantitative PCR was performed using the two primer sets and chromatin DNA contained in the immunoprecipitates. Values from IgG-immunoprecipitated samples were arbitrarily set to 1.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: In Vitro, In Vivo, Membrane, Labeling, Incubation, Recombinant, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Administering L165041 to mice increases hepatic Lpcat3 mRNA and LPCAT enzyme activity along with elevations of Acsl4 and other PPARδ-target genes. Male C57BL/6 mice were gavaged daily with 40 mg/kg L165041 (n = 5) or vehicle (n = 5). After 7 days of treatment, the liver was excised, and the following were measured. A, all isoforms of Lpcat mRNAs. B, D, and F, qRT-PCR was conducted to determine the relative expression levels (mean ± S.E. (error bars), n = 5/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group, which was set at 1. C, total LPCAT activity (mean ± S.E., n = 5/group). ***, p < 0.001, compared with the control group. E, Western blotting analysis of hepatic ACSL4 protein levels.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Depletion of hepatic LPCAT3 expression in mice fed a chow diet attenuated PPARδ-mediated activation of hepatic genes in the FA metabolic pathway. Male C57BL/6J mice (n = 5 animals/treatment) were retro-orbitally injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised for the total LPCAT activity assay (A and B) and gene expression analysis (C). Statistical analysis was performed using Student's t test for the enzyme assay (A and B), and one-way ANOVA with Dunnett post hoc test was performed in gene expression analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Injection, Activity Assay, Gene Expression, Enzymatic Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Effects of hepatic LPCAT3 knockdown on serum and hepatic lipid levels of mice with or without L165041 treatment. Male C57BL/6J mice (5 mice/group) were injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised. Serum TG (A), TC (B), PL (C), and NEFA (D) were measured. Liver lipids were extracted and used to measure hepatic TG (E), TC (F), PL (G), and NEFA (H). Statistical analysis was performed using one-way ANOVA with Dunnett's post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Knockdown, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: qRT-PCR primer, EMSA probes, promoter cloning primers, and ChIP primer sequences

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Cloning

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 is required for PARP1-dependent necrosis after H2O2 treatment. (a) Left panel: WT MEFs were transfected with the vector control (VC) or Flag-FAF1 plasmids. At 36 h after transfection, cells were pretreated with vehicle (DMSO), zVAD-fmk (100 μM) or DPQ (30 μM) for 1 h and were then treated with 500 μM H2O2 for 6 h in presence of individual compounds. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. (b) Immunoblot analysis of FAF1 expression in immortalized Faf1+/+ and Faf1gt/gt MEFs. β-actin expression was used as an endogenous control. (c) Faf1+/+ and Faf1gt/gt MEFs were treated with the indicated concentrations of H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. Cell death was determined on the basis of LDH release (n=3). (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. Data (a, c–e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a, c–e) post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 translocates to the nucleus and interacts with PARP1 during oxidative stress. (a) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblotting with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytoplasmic marker) antibodies. (b) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then immunostained with the anti-FAF1 antibody. The nuclei were stained using propidium iodide (PI) and analyzed by confocal microscopy. (c) WT MEFs were transfected with the indicated combination of V5-PARP1 and Flag-FAF1 plasmids. At 36 h after transfection, the cells were untreated or treated with 500 μM H2O2 for 30 min. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (d) WT MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates then were immunoprecipitated with the anti-FAF1 antibody. The interactions were determined by immunoblotting with the indicated antibodies

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Western Blot, Marker, Staining, Confocal Microscopy, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes the catalytic activity of PARP1 during oxidative stress. (a) Left panel: WT MEFs were transfected with the indicated concentration of Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for 30 min. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (b) Left panel: WT MEFs were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (c) Left panel: Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 30 min and were then immunostained with the anti-PAR antibody. The nuclei were stained using PI and the cells were analyzed by confocal microscopy. (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). (f) Left panel: GST-FAF1 or GST was incubated with recombinant PARP1 (1 unit), β-NAD+ (100 μM) and damaged DNA for 10 min at room temperature. After the in vitro poly(ADP-ribosyl)ation reactions, the samples were subjected to immunoblot analysis. Right panel: the graph shows the results of densitometric analysis of PAR immunoblots (n=3). Quantified data (a–c, e, f) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a–c, e, f) post hoc analysis. **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activity Assay, Transfection, Concentration Assay, Western Blot, Staining, Confocal Microscopy, Incubation, Recombinant, In Vitro

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1-mediated PARP1 activation induces energy collapse, mitochondrial depolarization and AIF translocation during oxidative stress. (a and b) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 1 h. Depletion of intracellular energy was determined by measuring the levels of NAD+ (a; n=3) and ATP (b; n=3). (c) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 4 h. The cells were analyzed for mitochondrial membrane depolarization with a Muse analyzer (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 4 h and subsequently immunostained with the anti-AIF antibody. The nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) and the cells were analyzed by confocal microscopy. Data (a–c) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Translocation Assay, Membrane, Staining, Confocal Microscopy

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 mediates PARP1-dependent necrosis in SH-SY5Y cells during oxidative stress. (a) Left panel: SH-SY5Y cells were transfected with the VC or FAF1 plasmids. Thirty-six hours after transfection, the cells were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (b) Left panel: SH-SY5Y cells were transfected with scRNA or FAF1 siRNA. At 48 h after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (c) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblot analysis with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytosolic marker) antibodies. (d) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and then the cell lysates were immunoprecipitated with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies. (e) Left panel: SH-SY5Y cells were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). Quantified data (a, b, e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Western Blot, Expressing, Marker, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes dopaminergic neuronal cell death via PARP1 activation in an MPTP mouse model of PD. (a and b) The mice were administered four intraperitoneal injections of MPTP-HCl or saline at 2 h intervals. The mice were killed 24 h after the last injection. (a) Left panel: the subcellular localization of FAF1 was measured by immunofluorescence staining in TH-positive neurons of substantia nigra of mice that were treated with saline or MPTP. The nuclei were stained using DAPI and the tissues were analyzed by confocal microscopy. The white arrows indicate the presence of nuclear FAF1 in TH-positive neurons after MPTP treatment. Right panel: enlarged images of TH-positive neurons were taken from the white box in the merged images. (b) Brain lysates were prepared from the ventral midbrain of saline and MPTP-treated mice and were then subjected to immunoprecipitation with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies (n=3 per group). (c) The experimental scheme for panel d–f. The mice were injected unilaterally into the substantia nigra (SN) with adeno-associated virus type 1 (AAV1)-FAF1. Two weeks after AAV1-FAF1 injection, the mice were administered MPTP-HCl or saline, at 2 h intervals. The mice were killed 1 day and 7 days after the last MPTP injection and their brain tissues were prepared for western blot (WB) analysis or immunohistochemistry. (d) Left panel: at day 1 after the last MPTP injection, brain lysates were prepared from the ventral midbrain of the AAV1-FAF1-injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice and were then subjected to immunoblot analysis using the indicated antibodies. Right panel: the graphs show the results of densitometric analysis of PAR and FAF1 immunoblots in left panel (n=3 per group). (e and g) At day 7 after the last MPTP injection, the dopaminergic neurodegeneration was measured by histological analysis for TH-positive (e) and Nissl-positive cells (g) in SN of the AAV1-FAF1 injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice. Dashed lines represent a region of SN. (f) The graph shows the results of densitometric analysis of TH-stained neurons in e (n=5 per group). (h) The graph shows the counts of Nissl-stained cells in g (n=5 per group). Quantified data (d, f, h) are expressed as the mean±S.E.M. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Saline, Injection, Immunofluorescence, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Virus, Immunohistochemistry

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: (A) Reginal (7p22 locus) association of GWAS variants with plasma cholesterol levels shows that common SNP rs1997243 (purple diamond) and GPR146 p.Gly11Glu (red circle) are significantly associated with plasma TC levels in humans.

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques: Clinical Proteomics

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: (A-D) Changes of plasma TG (A and C) and TC (B and D) after Poloxamer-407 injection in Gpr146 whole body knockout mice (Gpr146+/+ vs Gpr146−/−, n=12–14 mice per group, by Student’s t-test) and liver-specific knockout mice (Alb-Cre- vs Alb-Cre+, n=8 mice per group, by Student’s t-test) fed chow.

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques: Clinical Proteomics, Injection, Knock-Out

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: (A and C) Plasma TC levels of Gpr146 whole-body knockout male (A) and female (C) mice lacking LDLR fed chow or western diet (WD) for 16 weeks (n=6–10 mice per group, by one-way ANOVA).

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques: Clinical Proteomics, Knock-Out, Western Blot

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: (A) Schedule of AAV-mediated knockdown of GPR146 in mice lacking LDLR.

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques: Knockdown

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: (A) GSEA enrichment plot of MAPK_Signaling gene set (KEGG pathway database) in liver of Gpr146+/+ mice and Gpr146−/− littermates (n=3 mice per group) upon 6-hour refeeding after a 16-hour fast.

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques:

Journal: Cell

Article Title: GPR146 Deficiency Protects Against Hypercholesterolemia and Atherosclerosis

doi: 10.1016/j.cell.2019.10.034

Figure Lengend Snippet: Key Resource Table

Article Snippet: To knock down hepatic Gpr146 in vivo , AAV8-scramble-shRNA (Vector Biolabs) or AAV8- Gpr146 -shRNA (Vector Biolabs) were administered to eight-week-old Ldlr −/− mice via retro-orbital injection, with a dose of 6 × 10 11 gc per male mouse and 4 × 10 11 gc per female mouse.

Techniques: Virus, Plasmid Preparation, Recombinant, Clinical Proteomics, Quantitation Assay, Microarray, Sequencing, Software

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Treatment of BH3-mimetic drugs in HCC1195 and HCC1438 cells. (a-c) Cytosol and mitochondrial proteins were extracted from HCC1195, HCC1438, CD3 + T, pulmonary fibroblast cells, and expression of Bcl-2 or Bcl-xL was determined using western blotting. Beta-tubulin was used as a positive marker for the cytosol, while COX IV was used as a positive marker for the mitochondrial fraction. (d) Cell death was measured following treatment with 3 nM ABT199, 19 nM A115463, or 15 µM ABT737 for 1 h, or incubation with 200 pg/µL granzyme B and 100 pg/µL perforin for 2 h. (e, f) The cells were fixed up to 5 h, and apoptosis were determined using ELISA kit. (g) Structures of ABT199 and ABT199-BODIPY. (h) Following treatment of synthetic ABT199-BODIPY, refractive index images of HCC1195 were enlarged at 28, 30, or 32 min, and ABT199-BODIPY were colored in green. (i) Quantified mean fluorescent intensity of the BODIPY signal in cells was measured at 512 nm. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from cytosol fraction, P < 0.05. # Significantly different from treatment of ABT199-BODIPY at 0 min, P < 0.05. COX IV: cytochrome c oxidase subunit IV; BH3: Bcl-2 homology 3; Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; ELISA: enzyme-linked immunoassay; BODIPY: boron dipyrromethene.

Article Snippet: Briefly, in six-well culture plates, 10 6 cells were plated and exposed to the Bcl-2 wild type plasmid (8768, RRID: Addgene_8768, Addgene, USA), Bcl-xL wild type plasmid (113458, RRID: Addgene_113458, Addgene, USA), Bcl-2 CRISPR plasmid (sc-400025-KO-2, Santacruz, USA), Bcl-xL CRISPR plasmid (sc-400170, Santacruz, USA) or control plasmid (sc-418922, Santacruz, USA) solution for 8 h at 37 °C in a CO 2 incubator.

Techniques: Expressing, Western Blot, Marker, Incubation, Enzyme-linked Immunosorbent Assay, Refractive Index

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Role of expression of Bcl-2 or Bcl-xL in extracellular mitochondria on lung cancer cells. (a, b) CD3 + T cells treated with Bcl-2 CRISPR-plasmid, Bcl-2 wild type (WT)-plasmid, Bcl-xL CRISPR-plasmid, or Bcl-xL wild type-plasmid. The overexpressed (OE) and knocked out (KO) indicate overexpress and knock out. Mitochondrial proteins were extracted from CD3 + T cells and expressions of Bcl-2, Bcl-xL, or COX IV were determined using western blotting. (c) Apoptosis was determined using ELISA kit. (d) CD3 + T cells, KO-CD3 + T cells, or OE-CD3 + T cells was incubated with HCC1195 or HCC1438 cells for 5 h. Apoptosis was induced following combinations of recombinant 100 pg/µL of perforin and 200 pg/µL of granzyme B. Cell images were rendered (e) or quantization of internalization of mitochondria (f) was determined by tomographic phase microscopy. (g, h) Apoptosis was determined using ELISA kit. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from treatment of negative control plasmid, P < 0.05. # Significantly different from treatment of CRISPR plasmid, P < 0.05. @ Significantly different from all other groups, P < 0.05. Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; COX IV: cytochrome c oxidase subunit IV; CRISPR: clustered regularly interspaced short palindromic repeats; ELISA: enzyme-linked immunoassay.

Article Snippet: Briefly, in six-well culture plates, 10 6 cells were plated and exposed to the Bcl-2 wild type plasmid (8768, RRID: Addgene_8768, Addgene, USA), Bcl-xL wild type plasmid (113458, RRID: Addgene_113458, Addgene, USA), Bcl-2 CRISPR plasmid (sc-400025-KO-2, Santacruz, USA), Bcl-xL CRISPR plasmid (sc-400170, Santacruz, USA) or control plasmid (sc-418922, Santacruz, USA) solution for 8 h at 37 °C in a CO 2 incubator.

Techniques: Expressing, CRISPR, Plasmid Preparation, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Microscopy, Negative Control

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Mitochondria of immune cells deficient in Bcl-2 and Bcl-xL are related to cytochrome C-related apoptotic signals. (a-f) CD3 + T cells was incubated with HCC1195 or HCC1438 cells for 5 h. Apoptosis was induced following combinations of perforin and granzyme B. Proteins were extracted from HCC1195 or HCC1438 cells, and expression of Bid, Bcl-2, Bcl-xL, cytochrome C or GAPDH were determined using western blotting. (g) Caspase-3 activity was determined using ELISA kit. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from treatment of perforin, P < 0.05. # Significantly different from incubation of CD3 + T cells, P < 0.05. Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme-linked immunoassay; KO: knocked out.

Article Snippet: Briefly, in six-well culture plates, 10 6 cells were plated and exposed to the Bcl-2 wild type plasmid (8768, RRID: Addgene_8768, Addgene, USA), Bcl-xL wild type plasmid (113458, RRID: Addgene_113458, Addgene, USA), Bcl-2 CRISPR plasmid (sc-400025-KO-2, Santacruz, USA), Bcl-xL CRISPR plasmid (sc-400170, Santacruz, USA) or control plasmid (sc-418922, Santacruz, USA) solution for 8 h at 37 °C in a CO 2 incubator.

Techniques: Incubation, Expressing, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Mitochondria of immune cells prevents tumor growth following activated immunity in vivo . (a) Beginning at 5 weeks of age, male mice were given intraperitoneally with anti-IgG2a, anti-PD-1 (10 mg/kg), CD3 + T cells (1 × 10 6 cells), or KO-CD3 + T cells (1 × 10 6 cells) for 12 days. The arrow indicates the injection days. (b) Graph showing the effect of treatments on the population of activated CD69 + T cells using CD69 ELISA kit. (c, d) Bar graph or images of tumor volume change for mice. (e) Cellular internalization of mitochondria from immune cells promoted antitumor immune response through cytochrome C related apoptosis. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from all other groups, P < 0.05. ELISA: enzyme-linked immunoassay; KO: knocked out; Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; ELISA: enzyme-linked immunoassay; Ig: immunoglobulin; PD-1: programmed cell death 1.

Article Snippet: Briefly, in six-well culture plates, 10 6 cells were plated and exposed to the Bcl-2 wild type plasmid (8768, RRID: Addgene_8768, Addgene, USA), Bcl-xL wild type plasmid (113458, RRID: Addgene_113458, Addgene, USA), Bcl-2 CRISPR plasmid (sc-400025-KO-2, Santacruz, USA), Bcl-xL CRISPR plasmid (sc-400170, Santacruz, USA) or control plasmid (sc-418922, Santacruz, USA) solution for 8 h at 37 °C in a CO 2 incubator.

Techniques: In Vivo, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: (A) Ai14 mice were injected i.c.v. with AAV5-CMV-CRE.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Injection

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: (A) AAV5-CMV-Ins2 was injected into the lateral ventricles of C57BL6/J mice.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Injection

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: (A) Ins1−/− Ins2fl/fl mice were injected i.c.v. with AAV5-CMV-CRE.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Injection

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: Hypothalamuses of animals injected i.c.v. with either AAV5-CMV-GFP or AAV5-CMV-Ins2 were collected, and total RNA was processed for transcriptome analysis (bulk RNA sequencing).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Injection, RNA Sequencing Assay

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: (A) Photomicrographs depicting the expression of GCaMP6f (green) and hM3Dq-mCherry (red) in EChP cells 10 days after simultaneous treatment with AAV5-CMV-GCaMP6f and AAV5-TTR-hM3Dq-mCherry. Scale bar, 50 μM.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Expressing

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Virus, Plasmid Preparation, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Anatomical and Neurochemical Profiles of GABAergic Projection Neurons in the Mouse Inferior Colliculus

doi: 10.1101/2025.09.02.673871

Figure Lengend Snippet: Axonal fibers of GABAergic neurons from IC subregions projecting to the MGB. AAV2-hSyn1(S)-Flex-tdTomato-T2A-sypEGFP was injected into each IC subregion of the GAD67-Cre mouse. a) Synaptophysin-GFP-positive fibers from GABAergic neurons in the CNIC were distributed mainly in the MGv. a’-c’) The boundaries of each MGB subdivision were delineated based on PKCδ immunoreactivity. b) Synaptophysin- GFP-positive fibers from GABAergic neurons in the ECIC were distributed mainly in the POL, MGv, PIN/PP. c) Synaptophysin-GFP-positive fibers from GABAergic neurons in the DCIC were distributed mainly in the MGv. The injection sites are displayed in the Supplementary Information Figure S1. Scale bar: 0.5 mm

Article Snippet: To visualize axonal fibers from the IC, 0.2 μl of adeno-associated virus serotype 2 (AAV2)-hSyn1(S)-Flex-tdTomato-T2A-synaptophysin-fused enhanced GFP (sypEGFP) ( ) (RRID:Addgene_51509; 7.8 × 10 11 vg/mL purified at the National Institute for Physiological Sciences, Section of Viral Vector Development) was injected into the IC at the following coordinates: CNIC (bregma, −5.02 mm; lateral, 0.5 mm; depth, 1.0 mm from the surface at a 60-degree angle from the horizontal plane in the medio-lateral direction); ECIC (bregma, −5.02 mm; lateral, 1.2 mm; depth, 0.7 mm from the surface at a 60-degree angle from the horizontal plane in the medio-lateral direction); and DCIC (bregma, −5.02 mm; lateral, 0.2 mm; depth, 0.3 mm at a 60- degree angle from the horizontal plane in the medio-lateral direction).

Techniques: Injection

Journal: Cell reports

Article Title: Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

doi: 10.1016/j.celrep.2023.112291

Figure Lengend Snippet: Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and AAV1-EF1a-DIO-EYFP were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-c-Fos (9F6) Cell Signaling Technology Cat# 2250; RRID: AB_2247211 Rabbit polyclonal anti-PACAP-38 Peninsula Laboratories Cat# T-4473; RRID: AB_519166 Sheep polyclonal anti-FoxP2 R&D Systems Cat# AF5647; RRID: AB_2107133 Mouse monoclonal anti-CGRP Abcam Cat# ab81887; RRID: AB_1658411 Alexa Fluor 568-conjugated donkey anti-mouse Invitrogen Cat# A10037; RRID: AB_2534013 Alexa Fluor 594-conjugated donkey anti-sheep Abcam Cat# ab150180; RRID: AB_2716768 Alexa Fluor 568-conjugated goat anti-rabbit Invitrogen Cat# A11011; RRID: AB_143157 Alexa Fluor 647-conjugated donkey anti-rabbit Invitrogen Cat# A31573; RRID: AB_2536183 Bacterial and virus strains AAV-EF1ɑ-DIO-EYFP Addgene Cat# 27056-AAV1; RRID: Addgene_27056 AAV-EF1ɑ-DIO-eNpHR3.0-EYFP Addgene Cat# 26966-AAV1; RRID: Addgene_26966 AAV-EF1ɑ-double floxedhChR2(H134R)-EYFPWPRE-HGHpA Addgene Cat# 20298-AAV1; RRID: Addgene_20298 AAVrg-EF1ɑ-mCherry-IRES-Cre Addgene Cat# 55632-AAVrg; RRID: Addgene_55632 AAV-hSyn-double floxedhChR2(H134R)-EYFP Addgene Cat# 26973-AAV1; RRID: Addgene_26973 AAV-hSyn-eNpHR3.0-EYFP Addgene Cat# 26972-AAV5; RRID: Addgene_26972 AAV-CAG-FLEX-GCaMP6s-WPRE-SV40 Addgene Cat# 100842-AAV1; RRID: Addgene_100842 AAVrg-hSyn-EGFP Addgene Cat# 50465-AAVrg; RRID: Addgene_50465 AAV-hSyn-EGFP Addgene Cat# 50465-AAV5; RRID: Addgene_50465 pENN.AAV-hSyn-Cre-WPRE-hGH Addgene Cat# 105553-AAV1; RRID: Addgene_105553 AAV-hSyn-FLEx-mGFP-2ASynaptophysin-mRuby Addgene Cat# 71760-AAV1; RRID: Addgene_71760 rAAV8-CAG-mCherry-IRES-WGA-Cre UNC vector Core Cat# AV5901FG Chemicals, peptides, and recombinant proteins Normal Donkey Serum Sigma Cat# D9663 Triton X-100 Sigma Cat# X100 DAPI mounting solution Vectorlab Cat# H-1200 Retrobead (red) LumaFluor N/A (Continued on next page) 14 Cell Reports 42, 112291, April 25, 2023

Techniques: Injection, Expressing, Retrograde Tracing, Labeling, Patch Clamp, Two Tailed Test