(A) Schematic presentation of the Surf4 conditional allele. Blue rectangles represent exons and black line segments represent introns. Red triangles denote loxP sites. Expression of a Cre …
Uncropped and unedited gel shown in Figure 1B.
n=3 per genotype. Crossbars represent the mean read count in each sample.
(A) Serum PCSK9 levels measured by ELISA in Surf4fl/fl Alb-Cre+ mice and Surf4fl/fl Alb-Cre- littermate controls (n=6 per genotype). (B) Immunoblot for PCSK9 and HSP90 (loading control) in liver …
Uncropped and unedited blots shown in Figure 2.
n=4 per genotype. Crossbars represent the mean, p-values were obtained from a two-sided Student’s t-test.
(A) Body mass of control (Surf4fl/fl Alb-Cre-), heterozygous (Surf4fl/+ Alb-Cre+), and homozygous (Surf4fl/fl Alb-Cre+) mice at 2 months of age (n=6 per genotype). p-value was obtained by a one-way …
(A) Steady-state serum APOB levels in control and Surf4fl/fl Alb-Cre+ mice at 2 months of age (n=4 per genotype). (B) Representative immunoblot for APOB and HSP90 in liver lysates with and without …
Uncropped and unedited blots shown in Figure 3.
Mice were administered 3H-labelled triolein by oral gavage. Blood samples were collected over 4 hr and assayed for (A) glucose, (B) triglycerides, (C) non-esterified fatty acids (NEFA), and (D) 3H …
(A) Relative liver mass in control and Surf4fl/fl Alb-Cre+ mice presented as percentage of total body mass (n=11 per genotype). (B) Relative fat and lean mass in the livers of control and Surf4fl/fl …
Crossbars represent the mean. p-values were obtained from two-sided Student’s t-test.
(A) Normalized (Norm.) liver Surf4 mRNA levels in mice treated with scrambled siRNA (control) or varying concentrations of Surf4 targeting siRNA (n=5 per group). (B–D) Plasma PCSK9, cholesterol, and …
Uncropped and unedited blots shown in Figure 6—figure supplement 1.
Genotype(Expected) | Surf4fl/+ Alb-Cre-(25%) | Surf4fl/+ Alb-Cre+(25%) | Surf4fl/fl Alb-Cre-(25%) | Surf4fl/fl Alb-Cre+(25%) | p (χ2) |
---|---|---|---|---|---|
Mating | ♂ Surf4fl/fl Alb-Cre- X ♀ Surf4fl/+ Alb-Cre+ | ||||
61 (25.8%) | 71 (30.1%) | 60 (25.4%) | 44 (18.6%) | 0.096 | |
Mating | ♂ Surf4fl/+ Alb Cre - X ♀ Surf4fl/fl Alb-Cre+ | ||||
8 (18.6%) | 9 (20.9%) | 12 (27.9%) | 14 (32.6%) | 0.549 | |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (M. musculus) | Surf4fl mice (C57BL/6 J) | Wang et al., 2021b | ||
Strain, strain background (M. musculus) | Albumin-Cre mice (C57BL/6 J) | Adams et al., 2014 | JAX 003574 | |
Strain, strain background (M. musculus) | Surf4fl/fl Alb-Cre+ mice (C57BL/6 J) | This paper | ||
Strain, strain background (M. musculus) | SpCas9 mice | Platt et al., 2014 | JAX 026556 | |
Strain, strain background (M. musculus) | C57BL/6 J mice | Jackson lab | JAX 0006640 | |
Genetic reagent (include species here) | AAV-Cre-sgRNA | This paper | ||
Antibody | Anti-APOB - rabbit polyclonal | Fitzgerald Industries Internationa | 70 R-15771 | WB(1:1000) |
Antibody | Anti-PCSK9 – rabbit polyclonal | Abcam | ab31762 | WB (1:1000) |
Antibody | Anti-LDLR – rabbit monoclonal | Abcam | ab52818 | WB (1:1000) |
Antibody | Anti-HSP90 – rabbit monoclonal | Cell Signaling Technology | 4877 | WB (1:1000) |
Antibody | Anti-SURF4 – rabbit polyclonal | Wang et al., 2021b | WB (1:1000) | |
Antibody | Anti-APOA1 – rabbit polyclonal | Fitzgerald Industries Internationa | 70 R-15769 | WB (1:1000) |
Antibody | Anti-Albumin – mouse monoclonal | Proteintech | 66051 | WB (1:1000) |
Antibody | Anti-Tubulin – rabbit polyclonal | Proteintech | 10094–1-AP | WB (1:1000) |
Antibody | Anti-GAPDH – rabbit monoclonal | Abcam | ab181602 | WB (1:1000) |
Sequence-based reagent | Primers | IDT | Sequences are listed in Supplement File 1 | |
Sequence-based reagent | CRISPR-gRNA | IDT | Sequences are listed in Supplement File 2 | |
Sequence-based reagent | SURF4-targeting siRNA | This paper | Sequences are listed in Supplement File 3 | |
Commercial assay or kit | Cholesterol assay kit | SB-1010–225 | Fisher Scientific | |
Commercial assay or kit | PCSK9 ELSIA | MPC900 | R&D Systems | |
Commercial assay or kit | APOB ELISA | ab230932 | Abcam | |
Commercial assay or kit | Triglycerides assay | 10010303 | Cayman Chemical | |
Commercial assay or kit | RNeasy Plus Mini Kit | 74134 | Qiagen | |
Commercial assay or kit | Power SYBR Green PCR Master Mix | 4367659 | Invitrogen |
Primer sequences used in this study.
Guide RNA (gRNA) sequence used for AAV-CRISPR mediated hepatic Surf4 inactivation.
siRNA sequences used to target hepatic Surf4 in this study.
DESeq2 output for differential gene expression analysis of RNA-seq data.