Journal: bioRxiv

Article Title: Cardiac REDD1 alters glucose and fatty acid metabolic gene expression via an mTORC1-independent, PPARα-dependent mechanism and drives hypertrophic growth

doi: 10.64898/2026.03.16.710895

Figure Lengend Snippet: A-D. Hearts of adult (8-14-week-old) male and female mice of the indicated genotypes were harvested and subjected to total RNA extraction and qPCR for the indicated genes. (A-B) n=9,11 ( Redd1) , n=12,12 ( Pdk1, Pdk2, Pdp1 ), n=10,12 ( Pdk3 ), and n=11,12 ( Pdk4, Pdp2 ), unpaired t test, 2-way ANOVA. (C-D) n=3,3,3,4 ( Redd1, Pdk1, Pdk2, Pdk3, Pdp1, Pdp2 ) and n=3,3,3,3 ( Pdk4 ), 1-way ANOVA, 2-way ANOVA. E-J. Hearts of adult (8-14-week-old) male and female mice of the indicated genotypes were harvested, lysed, and subjected to western blotting with the indicated antibodies. Signals were quantified with densitometry, normalized to total PDH, and plotted. (E-G) n=10,19 (pPDH (Ser293), pPDH (Ser300)), unpaired t test. (H-J) n=5,6 (pPDH (Ser293), pPDH (Ser300)), unpaired t test. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. M = marker.

Article Snippet: 2 μg RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) and the QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific) for the following genes: 18S (Mm03928990_g1), Redd1 (Mm00512504_g1), PDK1 (Hs05380290_s1), Pdk1 (Rn00587598_m1), PDK2 (Hs04965351_m1), Pdk2 (Rn00446679_m1), PDK3 (Hs03878443_s1), Pdk3 (Rn01424337_m1), PDK4 (Hs01037712_m1), Pdk4 (Rn00585577_m1), PDP1 (Hs01081518_s1), Pdp1 (Rn01437077_m1), PDP2 (Hs01934174_s1), Pdp2 (Mm02526496_s1), ACSL1 (Hs00960561_m1), or Nppb (Mm01255770_g1).

Techniques: RNA Extraction, Western Blot, Marker

Journal: Nature communications

Article Title: VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism.

doi: 10.1038/s41467-017-01327-4

Figure Lengend Snippet: Fig. 5 VSIG4 triggers PDK2 expression in macrophages. Macrophages from WT and Vsig4−/−mice were collected. a qRT-PCR detection of 4 Pdk isoforms in BMDMs. b Western blot analyzing PDK2, p-PDH-E1αs300, p-PDH-E1αs293, and total PDH. c The location of p-PDH-E1αs300 in mitochondria was analyzed by immunofluoresence double staining, scale bar = 20μm. d Western blot of PDK2, p-PDH-E1αs300, p-PDH-E1αs293 in liver tissues at 0 h and 48 h PI. RAW264.7 cells were transfected to expression of Vsig4, and cells were further treated with LPS (2 μg/ml), e Western blot analysis of PDK2 and p-PDH- E1αs300. f PDH activity analysis, n = 6 per group. The expression of Pdk2 in RAW264.7 cells was silenced by shRNA or enhancing Pdk2 expression by lentivirus infection. g Seahorse analysis of OCR after 2 h of LPS treatment (up), and basal and maximal OCR of the indicated conditions was plotted in bar graphs (down), n = 5 per group. h Flow cytometric assay of mtROS secretion after LPS administration. i ELISA of IL-6 and TNF in cultured supernatants, n = 4 per group. j Flow cytometric assay of LPS-caused CD40 expression at 6 h. Error bar, s.e.m. *p < 0.05,**p < 0.01, ***p < 0.001 and NS, p > 0.05 (Student’s t-test). Data are representative of three independent experiments

Article Snippet: The mouseVsig4 (NM_177789) cDNA ORF clone (#MR203780) and Pdk2 (NM_133667) cDNA ORF clone (#MG206400) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Double Staining, Transfection, Activity Assay, shRNA, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature communications

Article Title: VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism.

doi: 10.1038/s41467-017-01327-4

Figure Lengend Snippet: Fig. 6 VSIG4 promotes PDK2 expression through activating PI3K/Akt–STAT3. a Western blot of Akt and p-Aktser473 expression. b RAW264.7 cells were infected with different Vsig4 deletion constructs, cells were further treated with LPS (2 μg/ml), the expression of p-Aktser473 was analyzed by western blot. c Western blot of p-Aktser473 and PDK2 in VSIG4+RAW264.7 cells with mutation of Ser273, Ser276, Thr270, and Thr274 to Ala. VSIG4+RAW264.7 cells were treated with d the Akt inhibitor MK-2206, e the PI3K inhibitor Ly294002, and the expression of Akt, p-Aktser473, PDK2, and p-STAT3 was detected by western blot. f VSIG4+RAW264.7 cells were treated with the MK-2206, and the expression of STAT3/p-STAT3 was analyzed by western blot. g Western blot of p-STAT3/STAT3 and PDK2 in LPS-activated VSIG4+RAW264.7 cells followed with STAT3 inhibitor, S3I-201 (100 μM) treatment for 24 h. h Western blot of PDK2 in Stat3 silenced VSIG4+RAW264.7 cells. i The enrichment of p-STAT3 in Pdk2 gene promoter region was detected by ChIP-qPCR assay. j Human VSIG4+THP-1 cells were treated with microbeads-C3b (20 μg/ml) in the presence of LPS (2 μg/ml), and the expression of PDK2 was detected by western blot. Moreover, C3−/−BMDMs were transfected to overexpress VSIG4, and cells were further treated with LPS (2 μg/ml) for an additional 3 h, and the expression of PDK2 was detected by western blot. Error bar, s.e.m. *p < 0.05, ***p < 0.0001 and NS, p > 0.05 (Student’s t-test). Data are representative of three independent experiments

Article Snippet: The mouseVsig4 (NM_177789) cDNA ORF clone (#MR203780) and Pdk2 (NM_133667) cDNA ORF clone (#MG206400) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Infection, Construct, Mutagenesis, ChIP-qPCR, Transfection

Journal: Nature communications

Article Title: VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism.

doi: 10.1038/s41467-017-01327-4

Figure Lengend Snippet: Fig. 8 Forced overexpression of Vsig4 improves MHV-3-induced hepatitis. C57BL/6 WT mice were infected with lentivirus (107 PFU/mouse) to induce the expression of Vsig4 in vivo, these mice were further infected with MHV-3 at day 6. a Liver Vsig4 gene transcription was analyzed by qRT-PCR at day 6, n = 5 per group. b Western blotting for PDK2, p-PDH-E1αs300, FGL2, and proinflammatory cytokines TNF, IL-6 and IL-1β in liver tissues at 72 h of MHV-3 infection, n = 4 per group. c The architecture of the liver tissues at 72 h of infection was compared by H&E staining, scale bar = 20 μm, n = 5 per group. d The survival was monitored for a total of 20 days. Error bar, s.e.m. a *p < 0.05 was analyzed by Student’s t-test, and d was analyzed by log-rank test. Data are representative of three independent experiments

Article Snippet: The mouseVsig4 (NM_177789) cDNA ORF clone (#MR203780) and Pdk2 (NM_133667) cDNA ORF clone (#MG206400) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Over Expression, Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: Overview of the antibodies used for immunoblotting. Abbreviations: Ab—antibody, Mo—mouse, O/N—overnight, Rb—rabbit. Cell Signaling Technology (Danvers, MA, USA), Novus Biologicals (Centennial, CO, USA), Abcam (Cambridge, UK).

Article Snippet: PDK2 , 46 , Novus Biologicals , NBP1-87307 , Rb , 1:800 , O/N , Bio-Rad , 1706515 , 1:8000 , 1 h.

Techniques: Western Blot